结核分枝杆菌肝素结合血凝素与人IL12融合基因重组耻垢分枝杆菌的构建及鉴定

目的构建表达肝素结合血凝素与人IL12融合基因的重组耻垢分枝杆菌并对其进行鉴定。方法通过RT-PCR从人淋巴细胞获得hIL12基因模板,采用PCR技术克隆hIL12的P40和P35两个亚基及结核分枝杆菌的HBHA基因,通过酶切鉴定及DNA测序证实连接的基因片段均正确后,构建分枝杆菌穿梭表达质粒pSMT3-HBHA-hIL12,并用电穿孔法将重组质粒转到耻垢分枝杆菌中。分别用HBHA单克隆抗体和hIL12多克隆抗体检测HBHA和hIL12蛋白在重组耻垢分枝杆菌中的表达,并测定重组耻垢分枝杆菌生长状态的改变。结果重组耻垢分枝杆菌构建成功,通过HBHA单抗和hIL12多抗进行免疫荧光法检测证实HBHA和hIL12均有表达,并且重组的耻垢分枝杆菌的生长速度比正常组有较大的提高。结论成功构建了表达结核分枝杆菌HBHA基因和人IL12的重组耻垢分枝杆菌,为构建一种结核病的候选疫苗提供了实验基础。

Construction and identification of recombinant Mycobacterium smegmatis which expressing fusion gene of heparin-binding haemagglutinin and human IL12

In order to obtain and identify the recombinant M.smegmatis（Ms） which expressing fusion gene of HBHA and hIL12,subunits of human IL12 gene,P40 and P35 gene were amplified by PCR from products of RT-PCR of lymphocytes separated from fresh blood.Meanwhile,the HBHA gene was amplified from H37Rv genome.After analyzing by enzymatic digestion and DNA sequence,human IL12 gene and HBHA gene were cloned into vector pSMT3 to constitute the recombinant plasmid pSMT3-HBHA-hIL12,which was then transfected into Ms by electroporation.The rMs was analyzed by immunofluorescence to confirm HBHA and hIL12 were expressed,and then the state of growth of rMs was detected.The recombinant Ms,which confirmed expressing HBHA and hIL12 by immunofluorescence,was constructed successfully,and the growth rate of rMs was faster than nomal Ms.The recombinant Ms expressing HBHA and hIL12 was constructed successfully,which laying a foundation for the study of new candidate TB vaccines.