摘要
试验旨在对单增李斯特菌临床分离株(LM90SB2)的LPXTG基序表面蛋白lmo0331基因进行克隆、序列分析和原核表达。根据GenBank中lmo0331基因序列设计特异性引物,利用PCR方法扩增LM90SB2 lmo0331基因,将扩增产物克隆到pMD19-T载体,测序后进行核苷酸序列和蛋白结构域分析;构建表达质粒pET32a-0331,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后,利用SDS-PAGE和Western blotting检测重组蛋白。结果显示,LM90SB2 lmo0331基因核苷酸序列与NTSN株、F2365株、LL195株及WSLC 1033株的同源性均为100.0%,与N2306株、02-1792株、H34株的同源性均为99.9%。LM90SB2 lmo0331基因全长1 956bp,ORF全长为1 857bp,共编码618个氨基酸;蛋白结构域预测结果显示,lmo0331蛋白具有NEL、LRR、IR、PulA、LPXTG结构域。SDS-PAGE结果可见约88ku重组蛋白带,Western blotting表明该蛋白可与小鼠抗His标签单克隆抗体发生特异性结合。本研究成功构建了原核表达载体pET32a-0331,实现了lmo0331融合蛋白的表达,为进一步研究lmo0331基因的功能及单克隆抗体的制备奠定了基础。
The experiment was aimed to clone lmo0331 gene of LPXTG motif fromListeria monocytogenes clinical isolate（LM90 SB2）,analyze the sequence and express it in prokaryotic system.Specific primers were designed according to the sequence of lmo0331 in GenBank.LM90 SB2 lmo0331 gene was amplified by PCR,its amplified product was cloned into pMD19-T vector and sequenced for nucleotide sequence and protein domain analysis.The expression plasmid pET32 a-0331 was transformed into E.coli BL21（DE3）competent cells.After induced by IPTG,the recombinant proteins were detected by SDS-PAGE and Western blotting.The results showed that the homologies of LM90 SB2 lmo0331 gene with NTSN,F2365,LL195 and WSLC 1033 strains were 100.0%,and the homologies with N2306,02-1792 and H34 strains were 99.9%.LM90 SB2 lmo0331 gene was 1 956 bp in length and 1 857 bp in ORF,encoding a total of 618 amino acids.Protein domain prediction showed that lmo0331 protein had NEL,LRR,IR,PulA and LPXTG domains.SDS-PAGE results showed about 88 ku recombinant protein band,and Western blotting result showed that the protein could specifically bind with mouse anti-His-tag monoclonal anti-body.In this study,the prokaryotic expression vector pET32 a-0331 was successfully constructed,and the expression of lmo0331 fusion protein was achieved,which laid the foundation for further study on the function of lmo0331 gene and the preparation of monoclonal antibodies.
作者
杜冬冬
张奇文
李红欢
钱凌霄
马勋
DU Dongdong;ZHANG Qiwen;LI Honghuan;QIAN Lingxiao;MA Xun(College of Animal Science and Technology,Shihezi University,Shihezi 832000,Chin)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第5期1170-1176,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31360614)
