编码弓形虫表面抗原P30基因的克隆及在E.coli中的表达

Cloning of the gene encoding P30 surface antigen from Toxoplasma gondii and expression in E.coli

目的 构建编码弓形虫RH株表面抗原P30基因重组表达质粒 ,初步观察P30基因在E coli表达。方法 将P30基因定向克隆到分支杆菌 -大肠杆菌穿梭表达质粒热休克蛋白 70 (hsp70 )起动基因的下游的多克隆位点 ,构建重组表达质粒pBCG -P30 ;采用亚克隆技术 ,将含P30和hsp70起动基因的复合片段 ,插入表达载体 pBK -CMV质粒 ,转化大肠杆菌DH5α ,在卡那霉素阳性LB培养基平板筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒 pBK -P30转化大肠杆菌 ,IPTG诱导表达后进行SDS -PAGE和Westernboltting分析。 结果 1)阳性重组质粒 pBCG -P30、pBK -P30经酶切和PCR鉴定 ,与预期的结果相符合。 2 )序列测定证实克隆的基因为编码P30抗原的基因。 3)P30基因在大肠杆菌诱导表达后获得4 5kDa融合蛋白 ,此抗原未被弓形虫高免兔血清识别。结论 成功构建编码弓形虫表面抗原P30重组表达质粒 ,并在大肠杆菌中获得表达 。

Aim To construct recombinant plasmids DNA encoding P30 surface antigen from Toxoplasma gondii and express in E coli DH Methods P30 gene from T gondii was cloned to the downstream of hsp70 promoter gene of E coli Mycobacteria shuttle plasmid,the recombinant shuttle expression plasmid,pBCG P30,was constructed A compound gene fragment of including P30 and hsp70 promoter gene was inserted into expression vector pBK CMV by subclone technique,then the recombinants were transferred into E coli DH5α and identified by restriction enzyme digestion and PCR Then the genetically engineered bacteria of including pBK P30 plasmids were induced by IPTG,the expression product was analyzed by SDS PAGE and Western bolting Results 1) The positive recombinant plasmids pBCG P30 and pBK P30 were identified by restriction enzyme digestion and PCR,in accordance with the expected results 2) Sequence determination analysis showed that the cloned gene was gene fragment coding P30 surface antigen from T gondii 3)Plasmid pBK P30 could express a specific 45kDa fusion protein in E coli DH5α,the fusion protein could not be recognized by immune rabbit serum Conclusion The expression plasmids which contain the gene fragment encoding P30 surface antigen from T gondii have been successfully constructed Plasmid pBK P30 can express specific fusion protein in E coli DH5α ,these results provided the basis for study of recombinant DNA vaccines anti Toxoplasma