单核细胞增生性李斯特氏菌溶血素基因克隆及原核表达载体构建

目的　构建单核细胞增生性李斯特氏菌 (Listeriamonocytogenes,LMO)溶血素基因重组表达质粒。 方法　本文采用PCR方法扩增出LMO 0 5 86株溶血素 (Hemolysin ,hly)基因 ,将其克隆到pMD18-T中 ,筛选带有插入片段的阳性质粒。经酶切和PCR鉴定 ,然后进行测序。继而用SalⅠ和XbaⅠ双酶切阳性质粒 ,目的片段定向插入pPROEXTMHTa中进行鉴定。结果　hly基因体外扩增产物大小约为 16 4 6bp。核苷酸序列鉴定后 ,其核苷酸序列与国外报道的LMOF6 789株、LMOF2 36 5株同源性分别为 99 70 %和 99 39%。推导出的氨基酸序列与其相应菌株比较 ,同源性分别为 99 82 %和 98 90 %。重组表达质粒经PCR及酶切鉴定表明为正确重组子。结论　在国内首次克隆到LMOhly全基因 ,并构建出含hly基因的原核表达载体。此项工作为进一步LMO溶血素基因的表达奠定了基础。

Cloning,sequencing of hly gene of Listeria monocytogenes(LMO) and its expression vectors construction

Objective To construct a recombinant plasmid containing Listeria monocytogenes (LMO)hemolysin(hly)gene.Methods A pair of primers were designed according to the published DNA sequence of LMO hly and were used to amplify gene for coding the best-characterized determinant of Listeria monocytogenes (LMO)by polymerase chain reaction(PCR).The purified PCR product was then cloned into pMD18-T vector.The recombinants were screened and identified by restriction analysis and PCR,and the cloned gene was sequenced.After inserting the double digested(SalⅠ/XbaⅠ)hly fragment into correspondingly expression plasmid pPROEX TMHTa,the recombinant plasmid was isolated and confirmed.Results The size of amplified hly gene was 1646bp.Nucleotide sequence analyses of the hly from the strain indicated that 0586 shared 99.70% and 99.39% identities with F6789(serotype 1/2b)and F2365(serotype 4b)respectively and the deduced amino acid sequences were 99.82% and 98.90% correspondingly.The correct recombinant plasmid has been proved by endonuclease analysis and PCR assay.Conclusion The full hly gene has been gained firstly in China,and the recombinant expression vector(pPROEX TMHTa—hly)has been successfully constructed.This work provides a basis of the hly protein expression.