吕氏泰勒虫TlSP基因多态性免疫区在毕赤酵母中的高效分泌表达

Efficient secretory expression of the polymorphic immunodominant region of TlSP gene of Theileria luwenshuni in Pichia pastoris

为研制具有完整抗原性和天然结构的吕氏泰勒虫表面蛋白TlSP（Theileria luwenshuni surface protein）,采用RT-PCR方法从吕氏泰勒虫裂殖子总RNA反转录获得TlSP基因的c DNA序列,将其多态性免疫区域克隆至毕赤酵母表达载体p PIC9K,构建重组质粒p PIC9K-TlSP。p PIC9K-TlSP经SalⅠ酶切线性化后,电转化导入毕赤酵母菌株GS115和KM71中,经遗传霉素G-418筛选后得到高拷贝菌株,对PCR鉴定为阳性的菌株进行甲醇诱导表达。对GS115和KM71两种菌株表达重组蛋白TlSP的效果进行评估,并对TlSP的反应原性进行了分析。结果表明,反转录获得的TlSP基因的c DNA序列长为888 bp,其中多态性免疫区域长为552 bp;筛选得到抗遗传霉素G-418（4.0 mg/m L）的GS115菌株5株,KM71菌株7株,经PCR鉴定为阳性的2种菌株均成功表达了分泌型重组蛋白TlSP,GS115菌株的蛋白表达效果较好;重组蛋白TlSP能与吕氏泰勒虫、尤氏泰勒虫、绵羊泰勒虫及环形泰勒虫阳性血清发生反应,而与巴贝斯虫、绵羊无浆体阳性血清无反应。本研究为深入研究TlSP蛋白在羊泰勒虫病免疫学诊断和亚单位疫苗中的应用奠定了基础。

The present study aimed to prepare Theileria luwenshuni surface protein（lib,） wltn com- plete antigenicity and natural structure. The total RNA was extracted from T. luwenshuni merozoites and the eDNA of T1SP gene was reversely transcribed using RT-PCR. The polymorphic immunodominant region of TISP gene was cloned into pPIC9K vector to form the recombinant plasmid pPIC9K-T1SP. The recombinant plasmid was linearized by Sal I and then transformed into Pichia pastoris GSll5 and KM71 by electropo- ration,and the multi-copy transformants with hyper-resistance to Geneticin G-418 were obtained by screening. The positive strains by PCR were induced by methanol. The effect of recombinant T1SP expres- sion in GSll5 and KM71 was evaluated,and the reactionogenicity of T1SP was analyzed. The result showed that the cDNA sequence of T1SP gene was 888 bp and its polymorphic immunodominant region was 552 bp in length;5 colonies in GSll5 and 7 colonies in ffMT1 resistant to 4.0 mg/mL Geneticin were obtained,and the recombinant protein T]SP was secreted in the above two strains. The effect of T1SP expression in 65115 was better than kM71. The results showed that the recombinant T1SP could react with the positive sera against T.luwensfiuni,T. uilenbergi,T, ovis and T. annulata,but not with the positive sera against Babesia spp. and Anapiasma ovis. This study laid the foundation for further research on the immunological diagnosis and the development of subunit vaccines of TISP protein in ovine and caprine theileriosis.