一株滑液囊支原体的分离鉴定及其磷酸丙糖异构酶(Tpi)和丙酮酸激酶(PK)抗体制备  

Isolation and Identifi cation of a Mycoplasma Synovialis Strain and Preparation of Its Triose Phosphate Isomerase(Tpi)and Lian Pyruvate Kinase(Pk)Antibodies

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作  者:连丽燕 巩新民 郭龙宗 李玉燕 蒋蔚[1] 陈兆国[1] 祁克宗[4] 魏晓峰 韩先干[1] LIAN Liyan;GONG Xinmin;GUO Longzong;LI Yuyan;JIANG Wei;CHEN Zhaoguo;QI Kezong;WEI Xiaofeng;HAN Xaingan(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Shanghai Laboratory Animal Research Center,Shanghai 200241,China;Shandong Yisheng Livestock&Poultry Co.,Ltd.,Yantai 264010,China;Anhui Agricultural University,Hefei 230036,China)

机构地区:[1]中国农业科学院上海兽医研究所,上海200241 [2]上海实验动物研究中心,上海200241 [3]山东益生种畜禽股份有限公司,烟台264010 [4]安徽农业大学,合肥230036

出  处:《中国动物传染病学报》2023年第5期55-63,共9页Chinese Journal of Animal Infectious Diseases

基  金:上海科委实验动物专项(18140900700)。

摘  要:磷酸丙糖异构酶(Tpi)和丙酮酸激酶(PK)是滑液囊支原体(MS)进行糖酵解的关键酶,牛支原体和鸡毒支原体的Tpi(tpiA基因编码)和PK(pyk基因编码)位于细胞膜和细胞质上,参与对宿主细胞的粘附,而MS的tpiA和pyk分别编码Tpi和Pyk,目前尚未见相关研究。本研究首先对临床分离的MS分离株的生长特性进行研究,然后使用Overlap PCR对tpiA和pyk进行点突变扩增,分别将其克隆至pET-28a载体并在BL21(DE3)中表达,用获得的TpiA和Pyk蛋白免疫新西兰兔制备多克隆抗体。结果表明:分离的MS分离株(MS-SD2020)最高生长滴定为109,在生长60 h时达到最高滴定;对tpiA和pyk的序列进行分析表明,其在不同MS中高度保守,并成功表达大小分别为35.28和63.36 kDa的TpiA和Pyk重组蛋白,免疫后制备的兔多克隆抗体效价分别为32000和2048000,本研究为后续开展MS的TpiA和Pyk功能研究提供参考。Triosephosphate isomerase(Tpi)and pyruvate kinase(PK)are the key enzymes for glycolysis of Mycoplasmasynoviae(MS).Tpi and PK of M.bovineand M.gallisepticumare located on the cell membranes and cytoplasms,respectively,which participate in adhesion to host cells.However,tpiA and pyk of M.synoviaehave not been reported yet.The growth characteristics of M.synoviumisolated from a chicken farm in Shandong province were investigated in the present study.Then the point mutations of TpiAand Pykwere amplified by overlap PCR method,which were respectively linked to pET-28a expression vector and expressed in BL21(DE3).The recombinant proteins of TpiA and Pyk were used to immunize New Zealand rabbits,respectively.The results showed that the highest growth titration of theM.synoviaeisolates MS-SD2020 was 109 at 60 h of growth.Sequence analysis ofTpiAandPykshowed that both were highly conserved in differentM.synoviaespecies.The recombinant proteins of TpiA and Pyk with the sizes of 35.28 kDa and 63.36 kDa were successfully expressed,and the rabbit serum antibody titers were 32,000 and 2,048,000,respectively.This study provided a reference for the further study on the function of TpiA and Pyk inM.synoviae.

关 键 词:滑液囊支原体 磷酸丙糖异构酶 丙酮酸激酶 抗体制备 

分 类 号:S858.31[农业科学—临床兽医学]

 

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