6株禽源滑液囊支原体分离株的分子生物学鉴定及MSPB蛋白原核表达  

Molecular Biology Identification of Six Mycoplasma synoviae Strains and Expression of MSPB Protein

作　　者：孙佳琳 司朵朵 王建东 李继东[1] 李生虎[3] 何生虎[1] SUN Jia-lin;SI Duo-duo;WANG Jian-dong;LI Ji-dong;LI Sheng-hu;HE Sheng-hu(College of Animal Science and Technology,Ningxia University,Yinchuan,Ningxia,750021,China;Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan,Ningxia,750002,China;Ningxia Vocational and Technical College,Yinchuan,Ningxia,750000,China)

机构地区：[1]宁夏大学动物科技学院,宁夏银川750021 [2]宁夏农林科学院动物科学研究所,宁夏银川750002 [3]宁夏职业技术学院,宁夏银川750000

出　　处：《动物医学进展》2024年第2期22-28,共7页Progress In Veterinary Medicine

基　　金：宁夏自然科学基金项目(2021AAC03249);宁夏银川市校企联合创新专项(2022XQ009);宁夏回族自治区科技创新团队建设项目(2022BSB03107)。

摘　　要：从分子水平对6株禽源滑液囊支原体(Mycoplasma synoviae,MS)分离株进行鉴定,探讨MS宁夏本地株膜蛋白MSPB的生物学功能。通过PCR技术扩增分离株的16S rRNA和MSPB基因序列,并构建分子进化树,对6株MS分离株进行同源性分析;用生物信息学工具对MSPB在蛋白水平进行分析,预测其亚细胞定位,探究MSPB的理化性质。构建pET-30a-MSPB重组表达载体,在大肠埃希氏菌BL 21(DE3)中进行表达,用镍柱亲和层析纯化并用蛋白质免疫印迹验证其反应原性。结果6株MS分离株在16S rRNA基因序列上与MS参考菌株具有同源性;MSPB等电点低,疏水性氨基酸占比较高;成功对MSPB进行表达,大小约为70 ku,纯化所得MSPB蛋白能与MS阳性血清特异结合。结果表明,6株菌株为MS,而MSPB具有反应原性,可以作为候选抗原应用于MS的诊断或防控。In order to identify 6 strains of Mycoplasma synoviae(MS)isolated from Ningxia at the molecular level and explore the biological function of the membrane protein MSPB,the 16S rRNA and MSPB gene sequences were amplified by PCR.The molecular evolutionary tree was constructed based on the 16S rRNA sequence,and the homologies of 6 MS isolates with other Mycoplasma reference strains were analyzed.The protein sequence of MSPB were analyzed by using bioinformatics tools to predict the sub-cellular localization and other potential functions,and the antigenicity was explored at the theoretical level.The recombinant expression vector pET-30a-MSPB was constructed and transformed into E.coli BL21(DE3)for expression.The protein was purified through Ni-chelating affinity chromatography and and its reactivity was verified by Western blotting.The results showed that the 6 MS strains had homology with different strains of MS in 16S rRNA gene sequence.The MSPB protein was acidic protein with low pI and high content of hydrophobic amino acids.The MSPB recombinant protein was successfully expressed with 70 ku and could specifically bind to MS positive serum.These results indicated that 6 strains all belong to MS,and MSPB has reactogenicity and can be used as a candidate antigen for the diagnosis and prevention of MS.

关 键 词：禽滑液囊支原体 16S rRNA MSPB蛋白 系统进化树 序列分析 反应原性 

分 类 号：S852.4[农业科学—基础兽医学]