Prokaryotic And Eukaryotic Expression Of Haemaphysalis Longicornis Serine Proteinase Inhibitor Gene And Its Structural Prediction

Haemaphysalis longicornis is one of the extremely widespread and important suck-blooding ectoparasites, which mainly infested cattle, sheep and other vertebrates. It can act as vector of many important pathogens of animals, for example virus, rickettsia, leptospira, bacteria, protozoa, mycoplasma, chlamydia, and hazard the husbandry severely. Anti-tick vaccination is among the best control strategies.Serine proteinase inhibitor (serpin) is a kind of regulators of serine proteinase, playing key roles in a variety of physiological functions such as blood coagulation, fibrinolysis, complement activation,inflammation,and tumor suppression. They are found widely in animals, plants, viruses, bacteria. Serpin in ticks was involved in host's defense response such as blood coagulation and cytokine activation. The ability of tick transmission pathogen was weakening obviously on serine proteinase and its inhibitor immunized hosts. Haemaphysalis longicornis serpin-2 was identified in haemolymph, which belonged to"concealed antigen", and used HLS2 as anti-tick vaccine antigens candidate molecule. HLS2 possessed the antithrombin enzymatic activity, possibly involved in regulating blood coagulation.A pair of specific primers was designed according to the HLS2 gene sequence published in GenBank. The HLS2 DNA fragment of 1164 bp was amplified by RT-PCR from unfed adult H. longicornis tick total RNA and cloned into pET-30a and transformed into E. coli JM109. The positive colonies were identified by restriction endonuclease digestion and sequencing. The pET-30a-HLS2 expression vector was transformed into E. coli BL21 (DE3) and induced by IPTG. The expression product was analyzed by SDS-PAGE and the expression condition was optimized. The recombinant protein was purified and identified by Western blotting. The results indicated that cloned HLS2 DNA fragment had 98% identity to HLS2 sequence in GenBank (Accession no. AB162827). A 47 kDa HLS2 fusion protein was expressed in E. coli mainly as the inclusion bodies form. The optimum expression of HLS2 fusion protein was obtained with 1.0 mmol/L IPTG for 7h at 28℃. Western blotting analysis showed the HLS2 fusion protein was recognized by the anti-H. longicornis serum of rabbit.The HLS2 DNA fragment was amplified by PCR and cloned into pPIC9k to construct the eukaryotic expression vector pPIC9K-HLS2. The recombinant was linearized with SalⅠand electroporated into Pichia pastoris cell GS115. The multi-copy recombinant strains were screened by G418 and was induced by methanol. The culture supernatant was analyzed by SDS-PAGE and Western blotting. The results showed that pPIC9K-HLS2 was constructed successfully, the high-resistance strains were obtained by GS115, the farget protein of about 49KDa was expressed in P. pastoris and had immunological activity.This study will lay the foundation for the further study of anti- H. longicornis vaccine.The secondary structure of the HLS2 and its lymphocyte epitopes were predicted and evaluated comprehensively by DNAStar molecular biology software. The result showed that the HLS2 coding protein had higher antigenic index and higher lymphocyte epitopes, which was consistent with of the higher antigenicity of HLS2 coding protein.The HLS2 coding protein had three potential glycosylation sites. They provided some guidance and reference to further study the structure and function of the HLS2 coding protein.