Arginine Kinase In Haemonchus Contortus: Cloning,Expression And Antigenic Characteristics Analysis

Haemonchus contortus is a kind of blood feeding parasitic nematode of ruminants. Infection with this parasite can cause large quantities of animal deaths, especially in lambs, which brought huge economic losses to the livestock industry. So far controling of H. contortus is almost based on the using of the anthelmintics. However, the growing emergence of resistant strains of H. contortus has resulted in the need to find new ways to prevent and control this parasite.Research progress had been made in the immunology research for H. contortus. However, no commercial vaccines were used to prevent the infection this parasite. More work should be done on exploring new antigens and futher studying the biological characters of the known proteins. In this article, the arginine kinase gene in H. contortus was cloned, expressed and purified, then the catalytic properties of the recombinant AK was analyzed. In this study, the effects of the recombinant AK on the proliferation and cytokine expression of the goat peripheral blood mononuclear cells (PBMCs) were detected, and the localizations of AK of H. contortus were analysed.1. Arginine kinase in H. contortus:cloning and expressionA gene fragment of 1104 bp was produced by RT-PCR, using a pair of primers based on the arginine kinase sequence of H. contortus. This fragment was cloned into pMD19-T vector, sequenced and analyzed. It was founded that the gene was 99% similar with the gene of H.contortus AK available in GenBank. Then the prokaryotic expression plasmid pET32/AK was constructed by subcloning the ORF of arginine kinase into the vector pET-32a(+). The recombinants were transformed into Escherichia coli BL21 and induced by isopropyl-B-D-thiogalactopyranoside (IPTG). SDS-PAGE analysis showed that the recombinant protein was expressed with the molecular weight of 60.3×103. The recombinant protein was purified with purification kit and refolded utilizing urea as solvent step by step, then analyzed by Western blot. Result of Western blot showed that the recombinant protein could be recognized by the serum from the goat infected with H. contortus.2. Catalytic properties of recombinant arginine kinase of H. contortusThe activity of arginine kinase was calculated according to the contents of phosphorus. The reactions were carried out at 10,20,25,30,35,40,50 and 60℃ separately, then at 30 "C, the optimum pH was determined with pH range 5.0-10.0. Finally, the absorbance of the mixture was measured at 660nm. Analysis of the activity of the recombinant protein indicated that the optimum reaction temperature was 30℃ and the optimum pH was 7.5 for this enzyme.3. Effects on cytokine expression in the goat PBMCs induced by the recombinant AKThe recombinant AK (0,10,20 and 40μg/mL) were incubated with the goat PBMCs separately, and the mRNA of different cytokines (IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β) were quantitied by Real Time PCR, and calculated by 2-△△Ct method. Results showed that the mRNA transcription levels of IL-17 in 40μg/mL group and 20μg/mL group were 9.45 and 2.69 times of the control group, respectively. The mRNA transcription levels of IFN-γ in 10,20 and 40μg/mL were 2.20,3.15 and 4.14 times than the control group. While the mRNA transcription levels of IL-2, IL-4, IL-10 and TGF-β had no significant difference.4. Effects of the recombinant AK of H. contortus on inducing the proliferation of goat PBMCsThe recombinant AK (0,10,20 and 40μg/mL) was incubated with goat PBMCs separately for 24 hours, at 37℃. Then the proliferaton index of these PBMCs was detected by the Cell Counting Kit-8. The result showed that in the group of ConA, the cell proliferation index was 2.43; In the group of 10μg/mL and 20μg/mL, the cell proliferation index were 1.68 and 1.39, while in the control group the index was 0.95. The results indicated that the recombinant AK could induce the proliferation of goat PBMCs.5. The localization of AK in adult H. contortusIn order to study the localization of AK in H. contortus, the whole frozen sections of adult worm of H. contortus were studied by immunohistochemical staining with anti-AK serum as the primary antibody.The result showed that AK was highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, and intestine, while it was absent in noncellular areas such as cuticle.