鸡毒支原体p24蛋白的原核表达及膜定位研究

Prokaryotic expression and membrane localization of p24 protein of Mycoplasma gallisepticum

为研究p24蛋白在鸡毒支原体中的定位,对p24基因序列进行分析,PCR扩增p24基因并构建原核表达载体,表达p24蛋白,制备多克隆兔抗血清,ELISA测定其效价,提取鸡毒支原体总蛋白、膜蛋白、胞浆蛋白通过Western blot进行亚细胞定位。结果显示:p24基因高度保守,全长576 bp,编码192个氨基酸;成功构建表达载体pCold-p24,经转化、诱导表达后获得重组蛋白rp24,大小约为35 kDa;ELISA检测兔抗rp24血清效价为1∶12800,说明rp24蛋白具有很好的免疫原性;Western blot显示rp24蛋白在膜蛋白免疫印迹条带较亮,在胞浆蛋白中条带弱,证明p24主要分布在膜表面,是鸡毒支原体的膜蛋白。试验结果为后续基因工程疫苗研制、诊断试剂盒研发、致病机理研究奠定基础。

To study the localization of p24 protein in Mycoplasma gallisepticum(MG),the p24 gene sequence was analyzed,the gene was amplified by PCR,and a prokaryotic expression vector was constructed to express the rp24 protein.Polyclonal rabbit antiserum was prepared and its titer was determined by ELISA.Total MG protein,membrane protein and cytoplasmic protein were extracted and the subcellular localization of the recombinant protein was performed by Western-blot.The results showed that the p24 gene was highly conserved,that it was 576 bp long,and it encoded 192 amino acids.An expression vector pCold-p24 was successfully constructed here,and a recombinant protein rp24 was obtained after transformation and it expression was induced with a size of about 35 kDa.The serum titer of the rabbit anti-rp24 detected by ELISA was 1∶12800,indicating that the rp24 protein possessed good immunogenicity.Western blot showed that the immunoblotting bands of the rp24 protein were bright in the membrane proteins and weak in the cytoplasmic proteins,suggesting that p24 was mainly distributed on the surface of the MG membrane,and that p24 was the membrane protein of MG.This study laid a foundation for vaccine development in future MG genetic engineering,for diagnostic kit development,and for research on pathogenic mechanism.