细粒棘球绦虫AgB8／1-AgB8／2重组嵌合抗原表达系统的构建

目的构建pET32a—AgBS／1-AgBS／2原核表达载体，并对其重组蛋自进行原核细胞表达。方法从细粒棘球绦虫原头蚴中提取总RNA，反转录生成cDNA，以此cDNA为模板，用基因特异性引物分别扩增EgAgBS／1和EgAgBS／2基因编码其分泌型多肽的片段，经测序后，以此两条基因片段为依据，人2r-合成EgAgBS／1-EgAgBS／2嵌合抗原编码核酸序列，将其克隆至pUCm—T载体，测序鉴定其正确性。通过对pUCm—T／AgBS／1-AgBS／2重组质粒进行双酶切，将获得的AgB8／1-AgBS／Z嵌合抗原编码核酸序列用定向克隆技术克隆至原核表达质粒pET32a上，测序鉴定插入片段E-确后，转化至E.coli BL21（DE3）Lys S，IPTG初步诱导表达pET32a-AgBS／1-AgBS／2重组嵌合蛋白。用SDS—PAGE电泳分析鉴定重组蛋白的表达水平。结果测序表明，AgBS／1-AgBS／2嵌合抗原编码核酸序列正方向插入至pET32a质粒。SDS-PAGE电泳分析显示，IPTG诱导后重组嵌合蛋白得到成功表达，在相对分子量约38kD处有表达条带。结论成功构建了pET32a—AgBS／1-AgBS／2原核表达质粒，并初步诱导表达出AgBS／1-AgBS／2嵌合重组蛋白，为进一步研究其免疫学特性奠定了基础。

Establishment of Echinococcus granulosus AgB8/1-AgB8/2 chimeric recombinant protein expression system

In order to construct the pET32a-AgBS/1-AgB8/2 chimeric antigen prokaryotic expression recombinant plasmid and the expression of its recombinant protein, the total RNA was extracted from protoscoleces of Echinococcus granulosus, and reverse transcribed into cDNA, the cDNA encoding mature form of EgAgB8/land EgAgB8/2 antigen were amplified by PCR using gene specific primers. Based on the both gene fragments, a nucleotide sequence encoding EgAgB8/1-EgAgB8/2 chimeric antigen were artificially synthesized after sequence confirmation. The synthesized nucleotide sequence encoding EgAgB8/ 1-EgAgBS/2 chimeric antigen were conformed by sequencing after cloning into pUCm-T vector, then the target sequence was directionally ligated into pET32a plasmid after double digestion with restriction enzymes for prokaryotic expression. The con structed recombinant plasmid pET32a- AgB8/1-AgB8/2 was transformed into E. coli BL21 （DE3） LysS, and the recombinant chimeric protein expression was induced by IPTG. The recombinant protein expression was analyzed by SDS-PAGE. Sequence analysis revealed that the nucleotide sequence encoding for AgB8/1-AgB8/2 chimeric protein was directionally cloned into pET82a plasmid. SDS-PAGE analysis confirmed that the recombinant chimeric protein AgBS/1-AgB8/2 fused with Trx was successfully expressed in E. coli BL21, with its relative molecule mass of about 38 kD. In this study, the recombinant plasmid pET32a-AgB8/1-AgBS/2 is successfully constructed and the recombinant chimeric protein EgAgB8/1-EgAgB8/2 was expressed.The results obtained in this study will provide a foundation for further study on its immune characteristics in the future.