结核杆菌HSP70在耻垢分枝杆菌中的表达及其免疫原性研究

Study on expression of TB HSP70 in M.smegmatis and its immunogenicity on mice

目的在重组分枝杆菌中表达编码人结核杆菌（ＴＢ）热休克蛋白７０（ＨＳＰ７０）的ＤｎａＫ基因，并观察其对小鼠的免疫效应。方法采用基因工程和免疫学技术将ＤｎａＫ基因及其两侧的表达调控区一起从质粒ｐＭＴ－７０中切出，经末端修饰后装入大肠杆菌－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中，构建成新的重组质粒ｐＢＣＧ－ＴＢ７０，并用以转化耻垢分枝杆菌及大肠杆菌，用Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的ＨＳＰ７０；以重组耻垢分枝杆菌分别经皮下及腹腔免疫小鼠，用淋巴细胞刺激指数（ＳＩ）反映细胞增殖能力，以ＮＯ法检测巨噬细胞吞噬活性，并检测血清中特异性抗ＴＢＨＳＰ７０的抗体。结果ＴＢＨＳＰ７０能在分枝杆菌中表达，表达量占菌体总蛋白量的１０％，不能在大肠杆菌中表达；重组耻垢分枝杆菌以１０６ＣＦＵ的剂量经皮下免疫小鼠后，可使小鼠脾淋巴细胞刺激指数（ＳＩ）和腹腔巨噬细胞吞噬活性增高（Ｐ＜０．０５），并能刺激机体产生特异性抗ＴＢＨＳＰ７０的抗体，腹腔免疫激发的抗体滴度较皮下免疫为低，而ＳＩ无明显改变。结论构建的表达质粒ｐＢＣＧ－ＴＢ７０能在耻垢分枝杆菌中表达ＨＳＰ７０，该重组菌具有较强的免疫原性。

Objective To express Dnak gene which codes human tubercle bacillus(TB) heat shock protein 70(HSP70) in M.smegmatis and determine its immunogenicity on mice. Methods Obtained from plasmid pMT 70,terminal modified DnaK gene had been cloned into shuttle vector pBCG2000 with its regulating sequences and a new plasmid pBCG TB70 with which Mycobacteria and E.coli were transformed had been constructed.Western blot was used for determining HSP70; lymphocyte stimulating index(SI)macrophage activity as well as the specific antibody to TB HSP70 were tested after immunization of mice with recombinant mycobacteria in a dose of 10 6 CFU by respective sc and ip. Results DnaK was expressed in recombinant M.smegmatis but not in E.coli and the production accounted for 10% of total bacterial proteins. Both SI and macrophage activity raised significantly ( P <0.05) and the antibody was detectable after sc injection while antibody was less and was no effect found on SI after ip injection. Conclusions DnaK gene for TB HSP70 in reconstructed plasmid pBCG TB70 can be expressed in the recombinant M.smegnatis which seems to be immunogenic crucially.