山羊支原体山羊肺炎亚种M17基因的原核表达及细胞定位研究

Prokaryotic expression of M17 gene of Mycoplasma capricolum subsp. capripneumoniae and its cellular localization

为分析山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae,Mccp)氨基肽酶M17的特征及细胞定位。参照GenBank中Mccp M1601株的M17基因序列,优化密码子,合成原核表达载体pET30a-M17,将鉴定正确的表达载体转化大肠杆菌BL21 (DE3),经IPTG诱导后获得融合表达蛋白;用其免疫新西兰兔,制备超免疫血清,并测定其效价和代谢抑制效果;通过Western-blot和i ELISA两种方法对M17在Mccp细胞内的分布进行了初步研究。结果显示,pET30a-M17重组蛋白在大肠杆菌中获得成功表达,大小约50 ku,与预期相符,密码子优化后的表达产物以可溶性和包涵体两种形式存在;免疫后的兔血清抗体效价达1∶80 000以上,说明M17重组蛋白具有较好的免疫原性和反应活性;M17多抗血清对Mccp无代谢抑制作用,但能够与Mccp的膜蛋白、胞浆蛋白及全菌蛋白发生特异性反应,表明M17蛋白在Mccp的细胞膜和细胞质中均有分布,但细胞膜中含量略高于细胞浆。M17的成功表达以及在Mccp中的细胞定位为其生物学功能研究奠定了基础,也为山羊传染性胸膜肺炎的诊断和疫苗研制提供了参考。

In order to analyze the characteristics and cellular localization of M17 protein of Mycoplasma capricolum subsp.capripneumoniae(Mccp).In this study,the M17 gene sequence of Mccp strain M1601 was retrieved from Genbank and optimized for prokaryotic expression.The recombinant plasmid of p ET30 aM17,which carry the M17 gene,was synthesized and transformed into Escherichia coli strain BL21(DE3).The recombinant BL21(DE3) was induced by IPTG to express M17 fusion protein.The expressed M17 protein was then purified and immunized to rabbits for preparation of hyperimmune sera.The distribution of M17 protein in Mccp cells were measured by Western-blotting and indirect ELISA,and metabolic inhibition test(MIT) was performed.The results showed that the recombinant M17 protein was successfully expressed in soluble form and inclusion bodies,with a molecular weight about 50 ku.The titer of antibody to M17 in the immunized rabbit serum was greater than 1∶80 000,which indicate that this protein has good immunogenicity and sero-reactivity.The polyclonal antibody to M17 binds the membrane protein,cytosol protein and whole protein of Mccp specifically in the Western-blotting,which suggest that the M17 protein is distributed in both membrane and cytoplasm of Mccp cell.The concentration of M17 protein in membrane is slightly higher than that in cytoplasm.The polyclonal antibody to M17 does not inhibit the metabolism of Mccp.This study lays the foundation for the biological function research on the protein M17,and also provides reference data to develop the vaccine and diagnostic tools for contagious caprine pleuropneumonia.