滑液囊支原体贵州株膜蛋白EF-Tu、pdhβ免疫原性分析

Antigenicity analysis of membrane proteins EF-Tu and pdhβ of Mycoplasmopsis synoviae Guizhou strain

为了研究鸡滑液囊支原体(Mycoplasma synoviae,MS)膜蛋白EF-Tu和pdhβ的免疫应答效果,试验针对目的基因设计特异性引物,经PCR扩增并构建原核表达载体,应用相关软件对其核苷酸和氨基酸序列进行生物信息学分析,对目的蛋白进行诱导表达并进行动物免疫试验。结果表明:PCR扩增EF-Tu和pdhβ基因得到特异性条带,大小为1185 bp和993 bp;成功构建了重组质粒pET32a-EF-Tu和pET32a-pdhβ;滑液囊支原体贵州分离株(MS-GZ-ZJ株)同其他流行株的EF-Tu基因同源性在99.7%~100%之间,pdhβ基因同源性在99.6%~100%之间,说明MS-GZ-ZJ株EF-Tu、pdhβ基因高度保守;EF-Tu、pdhβ蛋白的分子质量分别为43 ku和35 ku,是稳定的亲水蛋白;EF-Tu蛋白二级结构以α-螺旋、无规则卷曲和延伸链为主,pdhβ蛋白以α-螺旋和无规则卷曲为主。表达的蛋白大小与预期相符,EF-Tu蛋白主要为可溶性表达,pdhβ蛋白主要为包涵体表达;EF-Tu、pdhβ重组蛋白均能与MS阳性血清特异性结合。EF-Tu重组蛋白不能单独诱导细胞免疫应答,而pdhβ重组蛋白能刺激鸡群产生少量的白细胞介素4(IL-4),两个重组蛋白混合能诱导少量的IL-2和IL-4,但比疫苗组低很多;体液免疫应答效果不明确。说明单独使用经预测免疫原性好的蛋白质不一定能诱导动物机体产生良好的反应原性。

In order to study the immune response effect of Mycoplasmopsis synoviae protein EF-Tu and pdhβ, the experiment designed specific primers for the target genes, which were amplified by PCR and constructed a prokaryotic expression vector;bioinformatics analysis of its nucleotide and amino acid sequences was carried out using relevant softwares, and the target protein was induced to express and animal immunization test was carried out. The results showed that PCR amplification of EF-Tu and pdhβ genes yielded specific bands with sizes of 1 185 bp and 993 bp;recombinant plasmids pET32 a-EF-Tu and pET32 a-pdhβ were successfully constructed;the homology of EF-Tu gene between GZ-ZJ strain and other popular strains of Mycoplasmopsis synoviae Guizhou strain(MS-GZ-ZJ) was between 99.7% and 100%, and the homology between pdhβ gene and other popular strains was between 99.6% and 100%, which indicated that the EF-Tu and pdhβ genes of MS-GZ-ZJ strain were highly conservative. The molecular masses of EF-Tu and pdhβ proteins were 43 ku and 35 ku, respectively, which were stable hydrophilic proteins;the secondary structure of EF-Tu protein was dominated by α-helix, random coil and extended chain, and pdhβ protein was dominated by α-helix and random coil. The size of the expressed protein was consistent with the prediction. The EF-Tu protein was mainly expressed in soluble form, and the pdhβ protein was mainly expressed in inclusion bodies. Both EF-Tu and pdhβ recombinant proteins could specifically bind to MS-positive serum. EF-Tu recombinant protein could not induce cellular immune response alone, while pdhβ recombinant protein could stimulate chickens to produce a small amount of interleukin-4(IL-4);the combination of the two recombinant proteins could induce a small amount of IL-2 and IL-4, but much lower than the vaccine group;the effect of humoral immune response was unclear. The results suggested that the predicted protein with good immunogenicity might not be able to induce good reactogenicity in animal body when being used alone.