检索规则说明:AND代表“并且”;OR代表“或者”;NOT代表“不包含”;(注意必须大写,运算符两边需空一格)
检 索 范 例 :范例一: (K=图书馆学 OR K=情报学) AND A=范并思 范例二:J=计算机应用与软件 AND (U=C++ OR U=Basic) NOT M=Visual
作 者:赵勇[1] 张海[1] 赵善民[1] 伍静[1] 毛峰峰[1] 郭晓雅[2] 张彩勤[1] 师长宏[1]
机构地区:[1]第四军医大学实验动物中心,西安710032 [2]第四军医大学西京医院呼吸内科,西安710032
出 处:《中国人兽共患病学报》2011年第11期961-965,共5页Chinese Journal of Zoonoses
基 金:国家"863"专项课题(2007AA02Z473);国家自然科学基金(No.30972767);陕西省自然科学基金(SJ08C203;2007C224)联合资助
摘 要: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。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.
关 键 词:HBHA hIL-12 耻垢分枝杆菌 重组疫苗 融合蛋白
分 类 号:R378.9[医药卫生—病原生物学]
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在链接到云南高校图书馆文献保障联盟下载...
云南高校图书馆联盟文献共享服务平台 版权所有©
您的IP:3.138.112.77