镰形扇头蜱半胱氨酸蛋白酶基因的克隆与表达及表达产物免疫保护性分析

Subcloning and expression of gene encoding the cystein proteinase of Rhipicephalus haemaphysaloides tick and the immune protective analysis of its expressed products

目的在大肠杆菌中高效表达镰形扇头蜱半胱氨酸蛋白酶(cysteinase)CysA和CysB,并对表达产物进行免疫保护效果测定。方法将cysA和cysB基因亚克隆到pET-32a载体,转化感受态大肠杆菌BL21,在IPTG诱导下进行表达;表达产物免疫家兔,首次免疫抗原剂量免为300μg/兔,二次免疫和三次免疫均为150μg/兔。三次免疫后进行攻击蜱试验,观察免疫保护效果。结果在IPTG诱导下,重组质粒pET32a-cysA和pET32a-cysB在大肠杆菌中获得高效表达,产生Trx-cysA和Trx-cysB融合蛋白,这两种融合蛋白经纯化后免疫家兔,分别使成蜱的吸附率降至17%和17%,饱血率降至42%和22%。结论重组质粒pET32a-cysA和pET32a-cysB在大肠杆菌中高效表达,表达产物能诱导家兔产生一定程度的抗蜱保护性免疫力。

To subclone and express the gene encoding the cystein proteinase of Rhipicephalus haemaphysaloides tick and to analyze the immune protection of its expressed products, the cDNA of cys A and cysB genes were cloned into vector pET-32a, then transformed to E. coli BL21 cells and expressed by the induction with IPTG. The expressed products were purified and used as antigen to immunize rabbits in order to evaluate its immune protective effect, in which the rabbits in the vaccinated group were immunized with 300μg at first and 150 μg in the next two injections of the recombinant protein emulsified with Freund＇s complete adjuvant （FCA）, while those in the control group were injected with PBS emulsified with FCA, and the whole course of immunization consisted of triple injections at weeks 0, 2 and 4 respectively. The rabbits were challenged with R. haernaphysaloides ticks 2 weeks after the final injection, and the effect of immune protection was observed after challenging. It was found that the fusion protein Trx-CysA and Trx-CysB were highly expressed in E. coli in insoluble form and the inclusion bodies after induction by IPTG. The reduction rates of tick attachment in the vaccinated groups with both Trx-CysA and Trx-CysB were 17 % ; while the reduction in tick engorgement in the vaccinated groups with Trx-CysA and Trx-CysB were 42 % and 22 % respectively. It is evident from the above observations that the cysA and cysB genes can be highly expressed in E. coli after subcloning into vector pET-32a, and the fusion proteins Trx-CysA and Trx-CysB can significantly induce a certain degree of the anti-tick protective immunity.