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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 encoding Mycobacterium tuberculosis (TB) heat shock protein 70 (HSP70) in Mycobacterium tuberculosis and observe its immune effect on mice. Methods DnaK gene and its regulatory regions on both sides were excised from plasmid pMT-70 by genetic engineering and immunological techniques. After modification, the DnaK gene was inserted into E. coli-mycobacterium shuttle plasmid pBCG-2000 to construct The recombinant plasmids pBCG-TB70 were used to transform Mycobacterium smegmatis and E.coli. Western blot was used to detect the expression of HSP70. Mice were immunized subcutaneously and intraperitoneally with recombinant M. smegmatis, ) Reflect the cell proliferation ability, macrophage phagocytosis activity was detected by NO method, and the serum specific anti-TBHSP70 antibody was detected. Results TBHSP70 was expressed in mycobacteria and expressed in 10% of the total protein in E. coli. However, TBHSP70 could not express in E. coli. The recombinant M. smegmatis was immunized subcutaneously with 106CFU, Splenic lymphocyte stimulation index (SI) and peritoneal macrophage phagocytosis activity increased (P <0.05), and can stimulate the body to produce specific anti-TBHSP70 antibodies, antibody titers induced by intraperitoneal immunization is lower than the subcutaneous immunization, and SI no significant change. Conclusion The constructed expression plasmid pBCG-TB70 can express HSP70 in Mycobacterium smegmatis, which has strong immunogenicity.