**4. GST and immune system**

partially folded proteins, containing clusters of hydrophobic side chains accessible to sol‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Unbound ANS emission spectra showed a maximum at 530 nm that was blue shifted upon binding of the dye to the protein. Binding of ANS to BaGSTM as a function of GdmCl concen‐ tration showed one peak centered at 1.5 M and one peak as a function of urea concentrations centred at 3.5 M (Figures 7 a and b). ANS binding fluorescence of BaGSTM as a function of pH did not show any transition peak. However, the fluorescence intensity was increased as the pH decreased. The fluorescence intensity about 2000 fold higher at pH 2.0 compared with that at neutral pH (Figure 7 c). At the neutral pH, the fluorescence of ANS in the presence of BaGSTM is perfectly super imposable to that of ANS alone. At less than pH 3.8, ANS binds to BaGSTM showed a blue shift displacement with an enhancement of fluorescence intensity. Binding of the dye occurs at the dimer interface and unfolded GST does not bind ANS. This makes ANS an excellent probe to monitor changes at the packing of hydrophobic cores in protein which un‐ dergoes structural changes and has been broadly used to study the presence of monomeric in‐ termediates at the urea/GdmCl unfolding of several GSTs [38, 40-42]. ANS was also used to detect the presence of folding intermediates with hydrophobic patches such as the molten

15

**02468**

**[urea] M**

**b**

**Relative fluorescence intensity at 480 nm**

vent, is often observed in the presence of molten globules [23].

globule in penicillin G acylase [43] and apomyoglobin [44]. Running Title

**a**

**Relative fluorescence at 480 nm**

**0**

**500**

**1000**

**Fluorescence intensity at 480 nm**

**1500**

**2000**

1

Applications

278

2

7

and emission at 480 nm.

**0 1000 2000 3000 4000**

**[GdmCl]** mM

**1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0**

6 was added to the solution. Excitation was done at 380 nm and emission at 480 nm.

16 result is similar to that observed for sigma class GST in the presence of urea [46].

3 **Figure 7.** Variation of ANS binding fluorescence at 480 nm as a function of GdmCl concentrations 4 (a) or urea (b) or as a function of pH (c). The proteins (20 μg/ml) were equilibrated in buffer A at 5 room temperature in the presence of the indicated concentration of denaturant or pH. 100 μM ANS

**Figure 7.** Variation of ANS binding fluorescence at 480 nm as a function of GdmCl concentrations (a) or urea (b) or as a function of pH (c). The proteins (20 μg/ml) were equilibrated in buffer A at room temperature in the presence of the indicated concentration of denaturant or pH. 100 μM ANS was added to the solution. Excitation was done at 380 nm

 The unfolding results of the *R.* (Boophilus) *annulatus* (BaGSTM) demonstrate that the probes used (intrinsic fluorescence, excitation of tryptophan and tyrosine and ANS binding fluorescence) are differentially sensitive to various conformational states of BaGSTM. The presence of multiple nonsuperimposable transitions for this enzyme indicates that the two states unfolding mechanism is not applicable, and it is highly suggestion of the existence of at least two well-populated stable intermediates. Similar datra were reported demonstrating the existence of stable intermediates at the unfolding of GSTs [42, 45-48]. However, only one transition intermediate was detected using ANS binding fluorescence for BaGSTM in presence of different concentrations of GdmCl or urea. This

**pH**

Tick control is considered crucial all over the world to minimize the major drawbacks of tick infestations. The control strategies were adopted to use the acaricides which become less ef‐ ficient. Seixas et al. [49] has stated the alternative approaches used in tick control and are classified into four groups: (i) biological control by tick pathogens or predators [50]; (ii) habi‐ tat alterations by planting tick-killing or repelling vegetation [51]; (iii) immunological con‐ trol [52]; and (iv) development of tick-resistant breeds. They stated that although these methods have been proved to be theoretically valuable, most of them have been forsaken, since they did not afford acceptable cost/benefit ratios under field conditions, except for vac‐ cines.

Many types of chemical tick control were started since 1893 including the application of ar‐ senic acaricides, gamma BHC (organochlorine acaricide) and DDT. However, resistance to DDT was reported within 5 years of field application [53]. The toxicity plus the environmen‐ tal awareness and other factors led to the removal of the previously mentioned acaricides from the tick control market.

In early 1960's, Diazinon and other organophosphate acaricides were applied and in 1970's, Triatix was the first amidine to be registered for tick control. In the 1980's, pyrethroids (Flu‐ methrin and Permethrin) were also registered for tick control. The following table 2 shows the different acaricides used in tick control.

Along with the application of these chemicals with acaricidal properties as the predominant method of tick control throughout the world, resistance to the majority of the groups of chemicals had been evolved. Generally, the resistance may arise through several mecha‐ nisms including target sites, metabolic mechanism or reduced penetration. The target site re‐ sistance occurs when an allele of the gene coding for the target molecule is attacked by the acaricide. The penetration resistance occurs when an acaricide fails to penetrate the target individual. This type of resistance has not been reported in ticks. The third type of resistance mechanisms is the metabolic pathway which occurs through changes in the abilities of acari‐ cide detoxification by an organism [17]. Three enzyme families including cytochrome P450s

(115 individual members), esterases (81 individual members), and GSTs (39 individual members), are involved in the metabolic resistance mechanism.

Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especial‐ ly against schistosomiasis, fascioliasis and filarial parasites. However, several immunologi‐ cal studies were carried out to identify potential vaccines against helminth parasites including *Schistosoma mansoni* where the successful Sm28GST vaccine was developed by Capron et al. [62] and is in Phase II clinical trials. The injection of Sm28GST antigen elicited the production of immunoglobulins (especially IgE) and activation of eosinophils which could interfere with the function of parasitic GST [62]. Interestingly, the injection of Sm28GST in toxicity studies performed in dogs, rabbits and rats showed no system or local toxicity and no cross reactivity with rat or human GST [62]. Bushara et al. [63] and Morrison et al. [64] suggested that GST of *Schisotosoma spp*. and *Fasciola spp*. improved host immunity against these parasites. In this respect, GSTs were found to be up-regulated in response to rickettsial infection of *D. variabilis* ovaries [65]. They found that there is 0.25 fold increase in

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482 281

Previous studies correlated the role of GSTs in insect innate immunity with increased GST expression in response to infection-induced oxidative stress [66-70]. Increasing numbers of insect GSTs are being characterized due to their roles in insecticide detoxification. Dreher-Lesnick et al. [71] had cloned two GST variants and sequenced from the American dog tick; *D. variabilis* tick. Their structural analysis revealed that one of them belongs to the theta class (Figure 8) but no data is available about their biological activities. The secondary structure prediction using the DSSP prediction is shown in figure 9. Comparison of these two GST molecules with those of other species indicates that GST1 is related to the mammalian class theta and insect class delta GSTs, while GST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where GST1 and GST2 transcripts are found in the tick gut, with GST2 transcripts also present in the ovaries. Both *D. variabilis* GST transcripts are up-regulated upon tick feeding. The up-regulation of GST in this state is probably due to the stresses incurred during blood feeding. The authors could not rule out the possibility that up-regulation of GST in ticks may serve other purposes in‐ cluding cell protection from oxidative stress caused by infection with the intracellular bacte‐

*D. variabilis* serves as a host for an obligate intracellular cattle pathogen belongs to the genus Anaplasma; *Anaplasma marginale*. The developmental cycle of this pathogen begins in the gut cells of the host and the transmission to cattle occurs from the salivary glands during a second tick feeding. The *A. marginale* parasite has two stages occur within parasitophorous vacuole in the tick cell cytoplasm; the reticulated form (RF) which will transform to the in‐ fective dense form (DF). Kocan et al. [72] studied the characterization of the silencing effects of 4 different *D. variabilis* genes (separately) including the GST (DQ224235) on the develop‐ ment and infection levels of the *A. marginale*. They used the RNAi technology to silence these genes in male ticks and they showed that the *A. marginale* infection was inhibited both in tick guts after acquisition feeding and salivary glands after transmission feeding. *D. varia‐ bilis* ticks injected with GST dsRNA showed significant lower density of dense forms in guts after acquisition feeding. In general, the results of GST silencing demonstrate that GST is re‐

the mRNA expression of the GST gene.

rium Rickettsia.


**Table 2.** Different acaricide groups used in tick control.

Anti-tick vaccine development is focused on the identification, molecular cloning and in vi‐ tro production of proteins playing key putative roles in tick physiology, such as cell signal‐ ing, modulation of host immune response, pathogen transmission, embryogenesis, digestion, and intermediary metabolism [60]. Of the different antigens used as an anti-tick vaccine was the GST molecule. GST was of special interest to stimulate cattle immune sys‐ tem and their critical role in the metabolic resistance of acaricides. The immunological bases of using GSTs as vaccines may be derived from the hypothesis that parasites can survive within their hosts for a period of time despite the complex immune environment surround‐ ing them possibly accomplish this by adopting various immunomodulatory strategies, which include release of GSTs that counteract the oxidative reactive oxygen species (ROS) produced by the host activated cells and attach parasite cell membrane [61].

Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especial‐ ly against schistosomiasis, fascioliasis and filarial parasites. However, several immunologi‐ cal studies were carried out to identify potential vaccines against helminth parasites including *Schistosoma mansoni* where the successful Sm28GST vaccine was developed by Capron et al. [62] and is in Phase II clinical trials. The injection of Sm28GST antigen elicited the production of immunoglobulins (especially IgE) and activation of eosinophils which could interfere with the function of parasitic GST [62]. Interestingly, the injection of Sm28GST in toxicity studies performed in dogs, rabbits and rats showed no system or local toxicity and no cross reactivity with rat or human GST [62]. Bushara et al. [63] and Morrison et al. [64] suggested that GST of *Schisotosoma spp*. and *Fasciola spp*. improved host immunity against these parasites. In this respect, GSTs were found to be up-regulated in response to rickettsial infection of *D. variabilis* ovaries [65]. They found that there is 0.25 fold increase in the mRNA expression of the GST gene.

(115 individual members), esterases (81 individual members), and GSTs (39 individual

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Arsenite may bind to intracellular thiols, such as glutathione, and hence disrupting the ratio of reduced to oxidized glutathione leading to inhibition of cell division [54]

Interfere with the nerve conduction of ticks by affecting the ion channels, especially the voltage gated Na+ channels [55]

Suppress the enzyme acetyl-cholinesterase [56]. This group of acaricides is used in the form of phosphorothionates which are converted by the ticks into an active more toxic ingredient named "phosphate" or the "Oxon".

They inhibit the monoamine oxidase enzyme which is responsible for the metabolism of neurotransmitter amines in tick nervous system. They probably interact with octopamine receptors causing an increase in nervous activity causing detaching of ticks from the animal [57]

They interfere with nerve conduction [58]

It affects neural transmission. In ticks, ivermectin inhibits female engorgement by reduction of body weight [59]

members), are involved in the metabolic resistance mechanism.

DDT Gamma BHC Lindane Toxaphene

Diazinon Dichlorphos

> Amitraz Cymiazol

Cypermethrin Flumethrin Cyhalothrin Alphamethrin

Ivermectin

**Table 2.** Different acaricide groups used in tick control.

Arsenical Compounds Arsenic Trioxide

Chlorinated Hydrocarbons (Organochlorines)

Applications

280

Organophosphates

Amidines

Pyrethroides

Macrocyclic lactones (eg. Avermectin group)

**Type of Acaricide Examples Mode of Action**

Carbamates Propoxure Very similar to organophosphates

Anti-tick vaccine development is focused on the identification, molecular cloning and in vi‐ tro production of proteins playing key putative roles in tick physiology, such as cell signal‐ ing, modulation of host immune response, pathogen transmission, embryogenesis, digestion, and intermediary metabolism [60]. Of the different antigens used as an anti-tick vaccine was the GST molecule. GST was of special interest to stimulate cattle immune sys‐ tem and their critical role in the metabolic resistance of acaricides. The immunological bases of using GSTs as vaccines may be derived from the hypothesis that parasites can survive within their hosts for a period of time despite the complex immune environment surround‐ ing them possibly accomplish this by adopting various immunomodulatory strategies, which include release of GSTs that counteract the oxidative reactive oxygen species (ROS)

produced by the host activated cells and attach parasite cell membrane [61].

Previous studies correlated the role of GSTs in insect innate immunity with increased GST expression in response to infection-induced oxidative stress [66-70]. Increasing numbers of insect GSTs are being characterized due to their roles in insecticide detoxification. Dreher-Lesnick et al. [71] had cloned two GST variants and sequenced from the American dog tick; *D. variabilis* tick. Their structural analysis revealed that one of them belongs to the theta class (Figure 8) but no data is available about their biological activities. The secondary structure prediction using the DSSP prediction is shown in figure 9. Comparison of these two GST molecules with those of other species indicates that GST1 is related to the mammalian class theta and insect class delta GSTs, while GST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where GST1 and GST2 transcripts are found in the tick gut, with GST2 transcripts also present in the ovaries. Both *D. variabilis* GST transcripts are up-regulated upon tick feeding. The up-regulation of GST in this state is probably due to the stresses incurred during blood feeding. The authors could not rule out the possibility that up-regulation of GST in ticks may serve other purposes in‐ cluding cell protection from oxidative stress caused by infection with the intracellular bacte‐ rium Rickettsia.

*D. variabilis* serves as a host for an obligate intracellular cattle pathogen belongs to the genus Anaplasma; *Anaplasma marginale*. The developmental cycle of this pathogen begins in the gut cells of the host and the transmission to cattle occurs from the salivary glands during a second tick feeding. The *A. marginale* parasite has two stages occur within parasitophorous vacuole in the tick cell cytoplasm; the reticulated form (RF) which will transform to the in‐ fective dense form (DF). Kocan et al. [72] studied the characterization of the silencing effects of 4 different *D. variabilis* genes (separately) including the GST (DQ224235) on the develop‐ ment and infection levels of the *A. marginale*. They used the RNAi technology to silence these genes in male ticks and they showed that the *A. marginale* infection was inhibited both in tick guts after acquisition feeding and salivary glands after transmission feeding. *D. varia‐ bilis* ticks injected with GST dsRNA showed significant lower density of dense forms in guts after acquisition feeding. In general, the results of GST silencing demonstrate that GST is re‐

quired for pathogen infection of *D. variabilis* guts and salivary glands and IDE8 cells. It would suggest that GST may reduce the harmful effects of the metabolites, produced by the cellular oxidative stress, which may affect the development of the pathogen. Surprisingly, Kocan et al. [72] noticed that *A. marginale* infection was increased in the fat body cells in the GST silenced ticks.

Running Title

1 2

**Author details**

Yasser Shahein1

gy, Egypt

**References**

1959; 71 680-690.

zeus.few.vu.nl).

**. . . . . . . . . 210 . . . . . . . GST1 E P A I A F I K E R W A Q L K - - GST2 R G G F R S P T A H P G H G G A G**

, Amira Abouelella2

**. . . . . . . . . 10 . . . . . . . . . 20 . . . . . . . . . 30 . . . . . . . . . 40 . . . . . . . . . 50 GST1 M V A T L Y S V P A S T S C I F V R A L A R H I G F D L T V K Q L D F T K N E H L A E D Y L K L N P GST2 M A V E L Y N A T G S P P C T F V R V V A K K V G V E L T L H D L N L M A K E Q L N P E F V K L N P . . . . . . . . . 60 . . . . . . . . . 70 . . . . . . . . . 80 . . . . . . . . . 90 . . . . . . . . . 100 GST1 F H N V P T L D D G G F V V Y E S T T I A Y Y M L R K H A P E C D L Y P R S L E L R T R V D Q V L A GST2 Q H T V P T L N D N G F V L W E S R A I G M Y L V E K Y A P E C S L Y P K D V Q K R A T V N R M L F . . . . . . . . . 110 . . . . . . . . . 120 . . . . . . . . . 130 . . . . . . . . . 140 . . . . . . . . . 150 GST1 T V A T T I Q P K H F S F L R D T F C E N L K P T E G N M A A Y E E G V L K R L E L L I G A G P F S GST2 F E S G T M L P A Q M A Y F R P K W F K G - Q E P T A D L K E A Y D K A L A T T V T L L G D K K F L . . . . . . . . . 160 . . . . . . . . . 170 . . . . . . . . . 180 . . . . . . . . . 190 . . . . . . . . . 200 GST1 L G D T L T L G D L F I V S N L A V A L N T A A D P V K F P T L V D Y Y E R V K A A L P Y F E E I C GST2 C G D H V T L P D I G L A L H S G S S D W - G L R V R G P G Q V P P A Q G V L P A F Q E G L P R I R**

 **Figure 9.** *Dermacentor variabilis* theta Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respectively) 3-state (H, E C) secondary structure. The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no DSSP information available, or that no prediction was possible for that sequence. The

and Ragaa Hamed1

2 Department of Radiation Biology, National Centre for Radiation Research and Technolo‐

[1] Barnes M. M., James S. P., Wood P. B. The formation of mercapturic acids. 1. Forma‐ tion of mercapturic acid and the levels of glutathione in tissues. Biochemical Journal

[2] Booth J., Boyland E., Sims P. An enzyme from rat liver catalyzing conjugations with

[3] Freitas D.R., Rosa R.M., Moraes J., Campos E., Logullo C., Da Silva Vaz I. Jr., Masuda A. Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of *Boophilus microplus* (Acarina: Ixodidae). Comparative Biochemistry and Physiology. Part A Molecular In‐

**Figure 9.** *Dermacentor variabilis* theta Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respec‐ tively) 3-state (H, E C) secondary structure. The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no DSSP information available, or that no predic‐ tion was possible for that sequence. The conservation scoring was performed by PRALINE software (http://

7 conservation scoring was performed by PRALINE software (http://zeus.few.vu.nl).

1 Department of Molecular Biology, National Research Centre, Egypt

glutathione. Biochemical Journal 1961; 79 516-524.

tegrative Physiology 2007; 146(4) 688-694.

21

283

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482

Vaccination studies using tick proteins like GST from *Haemaphysalis longicornis* (Hl-GST) demonstrated the immunogenicity and antigenicity of this protein in bovines. Ultimately, immunization with GST protein triggered a partial immune response against *R. microplus* infestation in cattle, manifested mainly as a reduction of 7.9% in egg fertility, 53% in the number of fully engorged ticks and 57% overall efficacy ratio [73]. These data suggest that GST proteins have potential to be used as antigens in an anti-tick vaccine.

In conclusion, the phylogenetic analysis of the different cloned GST genes from different tick species indicates that numerous GSTs are present in the tick genome, which may or may not belong to different classes. These sequences are distributed in different tick organs including ovaries, gut and salivary glands. However, it is clear that protective immunity against tick infestation can be achieved; demonstrating that vaccination is a realistic unconventional ap‐ proach for tick control and GST would be a candidate.

1 2 3

20 Book Title

8 in figure 1. 9 10 11 **Figure 8.** *Dermacentor variabilis* Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respectively) amino acid conservation. The conservation scoring is performed by PRALINE software (http://zeus.few.vu.nl). The scor‐ ing scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment posi‐ tion. The color assignments are as in figure 1.

 **Figure 8.** *Dermacentor variabilis* Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respectively) amino acid conservation. The conservation scoring is performed by PRALINE software (http://zeus.few.vu.nl). The scoring scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position. The color assignments are as

2 3 **Figure 9.** *Dermacentor variabilis* theta Glutathione S-transferase GST1 and GST2 (DQ224235 and 4 AY241958, respectively) 3-state (H, E C) secondary structure. The Helix (H) structure is in red and 5 the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because 6 there is no DSSP information available, or that no prediction was possible for that sequence. The 7 conservation scoring was performed by PRALINE software (http://zeus.few.vu.nl). **Figure 9.** *Dermacentor variabilis* theta Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respec‐ tively) 3-state (H, E C) secondary structure. The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no DSSP information available, or that no predic‐ tion was possible for that sequence. The conservation scoring was performed by PRALINE software (http:// zeus.few.vu.nl).

#### 11 12 13 **Author details**

1

8 9 10

14

19

Running Title

quired for pathogen infection of *D. variabilis* guts and salivary glands and IDE8 cells. It would suggest that GST may reduce the harmful effects of the metabolites, produced by the cellular oxidative stress, which may affect the development of the pathogen. Surprisingly, Kocan et al. [72] noticed that *A. marginale* infection was increased in the fat body cells in the

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Vaccination studies using tick proteins like GST from *Haemaphysalis longicornis* (Hl-GST) demonstrated the immunogenicity and antigenicity of this protein in bovines. Ultimately, immunization with GST protein triggered a partial immune response against *R. microplus* infestation in cattle, manifested mainly as a reduction of 7.9% in egg fertility, 53% in the number of fully engorged ticks and 57% overall efficacy ratio [73]. These data suggest that

In conclusion, the phylogenetic analysis of the different cloned GST genes from different tick species indicates that numerous GSTs are present in the tick genome, which may or may not belong to different classes. These sequences are distributed in different tick organs including ovaries, gut and salivary glands. However, it is clear that protective immunity against tick infestation can be achieved; demonstrating that vaccination is a realistic unconventional ap‐

20 Book Title **. . . . . . . . . 10 . . . . . . . . . 20 . . . . . . . . . 30 . . . . . . . . . 40 . . . . . . . . . 50 GST1 M V A T L Y S V P A S T S C I F V R A L A R H I G F D L T V K Q L D F T K N E H L A E D Y L K L N P GST2 M A V E L Y N A T G S P P C T F V R V V A K K V G V E L T L H D L N L M A K E Q L N P E F V K L N P . . . . . . . . . 60 . . . . . . . . . 70 . . . . . . . . . 80 . . . . . . . . . 90 . . . . . . . . . 100 GST1 F H N V P T L D D G G F V V Y E S T T I A Y Y M L R K H A P E C D L Y P R S L E L R T R V D Q V L A GST2 Q H T V P T L N D N G F V L W E S R A I G M Y L V E K Y A P E C S L Y P K D V Q K R A T V N R M L F . . . . . . . . . 110 . . . . . . . . . 120. . . . . . . . . 130 . . . . . . . . . 140 . . . . . . . . . 150 GST1 T V A T T I Q P K H F S F L R D T F C E N L K P T E G N M A A Y E E G V L K R L E L L I G A G P F S GST2 F E S G T M L P A Q M A Y F R P K W F K G - Q E P T A D L K E A Y D K A L A T T V T L L G D K K F L . . . . . . . . . 160 . . . . . . . . . 170. . . . . . . . . 180 . . . . . . . . . 190 . . . . . . . . . 200 GST1 L G D T L T L G D L F I V S N L A V A L N T A A D P V K F P T L V D Y Y E R V K A A L P Y F E E I C GST2 C G D H V T L P D I G L A L H S G S S D W - G L R V R G P G Q V P P A Q G V L P A F Q E G L P R I R**

 **Figure 8.** *Dermacentor variabilis* Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respectively) amino acid conservation. The conservation scoring is performed by PRALINE software (http://zeus.few.vu.nl). The scoring scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position. The color assignments are as

**Figure 8.** *Dermacentor variabilis* Glutathione S-transferase GST1 and GST2 (DQ224235 and AY241958, respectively) amino acid conservation. The conservation scoring is performed by PRALINE software (http://zeus.few.vu.nl). The scor‐ ing scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment posi‐

GST proteins have potential to be used as antigens in an anti-tick vaccine.

proach for tick control and GST would be a candidate.

**. . . . . . . . . 210 . . . . . . . GST1 E P A I A F I K E R W A Q L K - - GST2 R G G F R S P T A H P G H G G A G**

1 2 3

8 in figure 1.

tion. The color assignments are as in figure 1.

GST silenced ticks.

Applications

282

15 16 Yasser Shahein1 , Amira Abouelella2 and Ragaa Hamed1

17 18 1 Department of Molecular Biology, National Research Centre, Egypt

20 21 2 Department of Radiation Biology, National Centre for Radiation Research and Technolo‐ gy, Egypt

## **References**

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[18] Eftink M. R., Helton K. J., Beavers A., Ramsay G. D. The unfolding of trp aporepres‐ sor as a function of pH: Evidence for an unfolding intermediate. Biochemistry 1994;

[19] Ptitsyn O. B. Molten globule and protein folding. Advances in Protein Chemistry

[20] Ptitsyn, O. B. Structures of folding intermediates. Current Opinion in Structural Biol‐

[21] Ausili A., Scire A., Damiani E., Zolese G., Bertoli E., Tanfani F. Temperature-induced molten globule-like state in human alpha1-acid glycoprotein: An infrared spectro‐

[22] Kuwajima K. The molten globule state as a clue for understanding the folding and

[23] Arai M., Kuwajima K. Role of the molten globule state in protein folding. Advances

[24] D'Alfonso L., Collini M., Baldini G. Does betalactoglobulin denaturation occur via an

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[26] Bychkova V. E., Dujsekina A. E., Fantuzzi A., Ptitsyn O.B., Rossi G. Release of retinol and denaturation of its plasma carrier, retinol binding protein. Folding and Design

[27] Dragani B., Cocco R., Principe D.R., Cicconetti M., Aceto A. Structural characteriza‐ tion of acid-induced intermediates of human glutathione transferase P1-1. Interna‐

[28] Goto Y., Calciano L. J., Fink A. L. Acid-induced folding of proteins. Proceedings of

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[30] Fink A. L. Molten globules. Methods in Molecular Biology 1995; 40 343-360.

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**Chapter 12**

**From Molecular Cloning to**

José Cantillo and Leonardo Puerta

http://dx.doi.org/10.5772/52821

results of this process are presented.

**1. Introduction**

Additional information is available at the end of the chapter

**Vaccine Development for Allergic Diseases**

Allergic diseases are manifested in susceptible individual by exposure to proteins named allergens that induce an immune response mediated by IgE antibody. Numerous allergens from different sources such as plants, insects, mites and mammals have been obtained as recombinant molecules by molecular cloning. These types of molecules have shown molecular, functional and immunological properties similar to the corresponding natural allergens and, therefore, could be used for in vitro and in vivo diagnosis test of allergy. An important step was done with the development of variants of allergens with reduced allergenicity and preserved immunogenicity, which paved the way toward its rational use in allergen specific immunotherapy to treat allergies. Few of the allergens cloned have been developed to a stage at which they are suitable for use in clinical studies. However, today the academic and scientific communities note a broad and important activity to offer in the near future preparations with enhanced clinical efficacy and safety. In this work, basic aspects and experimental and clinical

**2. Progress in the molecular cloning and production of allergens**

The molecular cloning has provided a practical and efficient way to obtain highly purified molecules for different purposes; in the biomedical sciences this is evident by the increasing amount of biological products, obtained by recombinant DNA technology, which are com‐ mercially available for diagnosis and treatment of different diseases, as well as the wide variety of reagents for basic research. The era of molecular cloning of allergen molecules was initiated in 1988 with the report of a cDNA clone coding for the allergen Der p 1 isolated from a cDNA

> © 2013 Cantillo and Puerta; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Cantillo and Puerta; licensee InTech. This is a paper distributed under the terms of the Creative Commons
