柔嫩艾美球虫杨凌株表面抗原SAG10基因的克隆与原核表达

Cloning and prokaryotic expression of the SAG10 gene encoding surface antigen 10 of Eimeria tenella Yangling strain

从柔嫩艾美球虫第二代裂殖子总RNA中扩增EtSAG10基因,与pGEM-T Easy载体连接后转化大肠杆菌(E.coli)DH5α,筛选阳性克隆,扩增不含N端信号肽的编码序列,分别插入表达载体pET-32a(+)和pMAL-c2X,转化至E.coliRosetta,以IPTG诱导表达。结果表明,pET-32a(+)-EtSAG10在E.coliRosetta中的表达产物约占菌体总蛋白的43%,融合蛋白分子质量约为47 ku,以包涵体形式存在;而pMAL-c2X-EtSAG10在E.coliRosetta中表达的重组蛋白为可溶性,表达产物约占菌体总蛋白的35%,融合蛋白分子质量约为69 ku。以表达的可溶性EtSAG10重组蛋白100μg/只肌肉注射免疫雏鸡,攻虫后以盲肠病变计分、盲肠卵囊数(OPG)、相对增重率和抗球虫指数(ACI)评价,免疫组相对盲肠卵囊产量为47.7%,抗球虫指数由86.79提高至152.13。提示,重组表达的EtSAG10可诱导雏鸡产生一定的抗球虫免疫保护。

The gene encoding EtSAG10（surface antigen 10 of Eimeria tenella） was amplified by RT- PCR,and cloned into pGEM-T Easy vector. EtSAGIO ORF fragment without the signal peptide sequence was sub-cloned,and inserted into the expression vector pET-32a（＋） and pMAL-c2X, respectively, for transforming Escherichia coli Rosetta. The cloned EtSAGIO sequence from E. tenella Yangling strain was highly similar to that from E. tenella H strain with a similarity of 98.9 %. The recombinant plasmid pET- 32a（＋）-EtSAG10 was able to be expressed as inclusion bodies in E. coli Rosetta under the induction of IPTG,and the molecular mass of the recombinant protein which accounted for 43 % of the total bacterial proteins was 47 ku as revealed by analysis using SDS-PAGE. However, the recon pMAL- c2X-EtSAGIO was able to be expressed as a soluble protein with a molecular mass of 69 ku in E. coli Rosetta induced by IPTG when being trigged at 20℃. The vaccinated chickens with 100 μg recombinant EtSAG10 per chicken were partially protected against the challenge of E. tenella occysts, which was evaluated using the weight gain,relative oocyst production and lesion score of the caeca, as well as the anti-coccidial index（ACI）. The results showed that EtSAG10 protein was a candidate antigen for the development of genetic vaccine against coccidiosis.