牛支原体pdhc基因的克隆、表达及其亚细胞定位

doi:10.16431/j.cnki.1671-7236.2018.09.004

摘要/Abstract

摘要：

试验旨在研究牛支原体（Mycoplasma bovis，M.bovis）武威株二氢硫辛酰胺转乙酰酶（PDHc-E2）基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因（登录号：CP002058.1）设计引物，应用PCR扩增获得牛支原体武威株pdhc基因，在测序及序列分析的基础上，应用Overlap PCR完成点突变后将其克隆至pET-28a（+）中，构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta（DE3）感受态细胞后经IPTG诱导获得融合蛋白，将纯化蛋白免疫新西兰兔制备多抗血清，应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示，牛支原体武威株pdhc基因CDS全长735 bp，编码244个氨基酸，与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致，与国际标准株PG45同源性为99.2%，与无乳支原体（M.agalactiae）同源性为90.9%~91.2%，与加利福尼亚支原体（M.californicum）ST6株的同源性仅为78.4%，基因序列非常保守；通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG，且完成点突变后的基因在大肠杆菌中成功表达，重组蛋白大小约为29 ku，主要以可溶性形式存在，iELISA结果显示，重组蛋白PDHc-E2具有较高的免疫原性，可刺激新西兰兔产生高水平的抗体，血清效价高达1：100 000；亚细胞定位结果表明，制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合，说明该蛋白在牛支原体细胞膜和细胞质中均有分布，为膜相关蛋白，但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。

关键词: 牛支原体; 二氢硫辛酰胺转乙酰酶; 原核表达; 亚细胞定位

Abstract:

To study the sequences characteristics of pdhc gene in M.bovis Wuwei strain and its location in M.bovis cells,the primers of pdhc gene were designed according to M.bovis HB0801 strain (accession No.:CP002058.1) in GenBank,and the pdhc gene of M.bovis Wuwei strain was amplified by PCR.Based on sequencing and gene analysis,the prokaryotic expression vector pET-pdhc was constructed,and then transformed into Escherichia coli Rosetta (DE3).After induced by IPTG,the M.bovis fusion protein PDHc-E2 was purified and New Zealand rabbits (Oryctolagus cuniculus) was immunized to prepare anti-serum against M.bovis rocombinant protein PDHc-E2.Subsequently,the distribution of PDHc-E2 in M.bovis cells was analyzed using iELISA and Western blotting.The results showed that the full-length sequence of pdhc gene CDS of M.bovis Wuwei strain was 735 bp and encoded 244 amino acids.It had 100.0% homology with the domestic M.bovis isolates,such as HB0801,Hubei-1,CQ-W70 and NM2012,and had 99.2% homology with the international standard strain PG45,the homology with Mycoplasma agalactiae (M.agalactiae) were 90.9% to 91.2%,the homology with the ST6 strain of Mycoplasma californicum (M.californicum) was only 78.4%,and the gene sequence was very conservative.By Overlap PCR,four TGA codons encoding tryptophan in this gene mutated to TGG;After point mutation the pdhc gene successfully expressed in Escherichia coli.The molecular weight of recombinant protein was approximately 29 ku,and it mainly existed in the form of soluble protein.iELISA results showed that recombinant protein PDHc-E2 had a high immunogenicity,and could stimulate New Zealand rabbit to produce high levels of antibodies,the iELISA titer of antiserum was approximately up to 1:100 000.The results of subcellular localization showed that the preparation of polyclonal antibody could bind specifically to recombinant protein PDHc-E2,total protein,membrane protein and cytoplasm of M.bovis,which indicated that the PDHc-E2 of M.bovis was distributed in both the membrane and the cytoplasm,and was a membrane-related protein,but the distribution in the cytoplasm was more than in the cell membrane.The results of this study laid a theoretical basis for further investigation on biological functions of M.bovis.

Key words: Mycoplasma bovis; dihydrolipoamide acetyltransferase; prokaryotic expression; subcellular localization

中图分类号:

S858.62

张懿, 邢小勇, 温峰琴, 包世俊. 牛支原体pdhc基因的克隆、表达及其亚细胞定位[J]. 《中国畜牧兽医》, 2018, 45(9): 2377-2385.

ZHANG Yi, XING Xiaoyong, WEN Fengqin, BAO Shijun. Cloning, Expression of pdhc Gene of Mycoplasma bovis and Its Subcellular Localization[J]. , 2018, 45(9): 2377-2385.

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