GLS/IL-12重组耻垢分枝杆菌的构建及其在小鼠体内的表达

Construction and expression of GLS/ IL-12 recombinant M. smegmatis

目的构建携带编码人天然颗粒溶素(GLS)和小鼠IL-12基因质粒(pZM03)的重组耻垢分枝杆菌,并经鼻黏膜免疫观察其在小鼠体内的表达。方法将人GLS和小鼠IL-12基因及分枝杆菌复制子OriM同时克隆进多启动子真核共表达载体pBudCE4.1中,得到穿梭质粒pZM03,以pZM03电转化耻垢分枝杆菌,再将该重组耻垢分枝杆菌滴鼻免疫Balb/c小鼠3次,第1次免疫后4周,处死小鼠,用免疫组化检测肺、脾组织中GLS表达,用ELISA检测血清IL-12和肺泡灌洗液特异性SIgA的水平。结果得到可表达GLS/IL-12重组耻垢分枝杆菌;在Balb/c小鼠肺、脾组织中均检测到重组耻垢分枝杆菌的分布,以及GLS的表达,并有血清IL-12水平升高和黏膜特异性SIgA的产生。结论成功构建GLS和IL-12修饰的重组耻垢分枝杆菌,该重组菌经鼻黏膜免疫小鼠后,可引起呼吸道黏膜特异性抗菌免疫,以及GLS和IL-12的体内表达,为新型疫苗的研究奠定了基础。

Objective To construct an eukaryofic coexpression plasmid encoding human granulysin （GLS） and murine IL- 12, and to observe the expression of recombinant Mycobacterium smegmatis carrying the coexpression plasmid in mice. Methods Coding sequences of human GLS, routine single chain IL-12, and OriM were simultaneously cloned into pBudCE4.1 for forming the shuttle-plasmid pBudCE4.1/GLS/ IL12/OriM（pZM03）. The shuttle-plasmid was transformed into M. smegmat/s and identified by PCR. Balb/c mice were immunized intranasally three times with recombinant M. smegmatis. Twenty-eight days after the first immunization, the expression of GLS in tissue, levels of IL- 12 in serum, and SIgA in bronchoalveolar lavnge fluid （BALF） were detected with immtmohistochemistry and ELISA, respectively. Results The new recombinant strain that could deliver pZM03 into mice was constructed suceessfully. The expression of GLS in tissue, the increased IL- 12 of in serum and the specific SIgA in BALF were found. Conclusion The specific anti-bacterium SIgA is generated. GLS and IL-12 are expressed in Balb/c mice after intranasal immunization with recombinant M. smegmatis carrying the plasmid encoding GLS and IL-12, which establish a foundation for new vaccine study.