多角体蛋白基因核苷酸序列对HBsAg(PreS2+S)在家蚕中表达的作用

The Effect of Nucleotide Sequence of Polyhedrin Gene on the Expression of HBsAg(PreS2+S) in Silkworm

应用PCR突变的方法 ,在乙肝病毒表面抗原 (HBsAg)前S2序列的 5’端融合了BmNPV多角体蛋白基因5’端的 12个碱基 ,获得了融合乙肝表面抗原中蛋白基因 (HBMp) ;通过同源重组将其插入到BmNPV基因组多角体启动子后 ,构建了重组杆状病毒BmPAK HBMp。用重组病毒BmPAK HBMp和BmPAK HBM(带有非融合乙肝表面抗原中蛋白基因 )感染家蚕细胞及蛹 ,对两种病毒的表达产物用ELISA进行了跟踪检测 ,结果表明融合蛋白比非融合蛋白HBsAg活性提高了 6 0 %～ 80 % ,作为HBsAg构成部分的PreS2抗原性提高了 8～ 10倍 ;并且HBsAg和PreS2 Ag表达的时相曲线不同 ,PreS2 Ag的表达量比HBsAg早 1～ 2d达到最大值。ELISA检测和Westernblotting分析表明 ,多角体基因序列的存在更有利于中蛋白全基因 (PreS2 +S)的表达。

The 12 bp of 5 end from the polyhedrin gene of BmNPV was fused to the 5 end of PreS2 by PCR mutation, the fusion HBV surface antigen middle protein gene (HBMp)was obtained. The recombinant virus by inserting HBMp into downstream polyhedron promoter of the BmNPV genome was constructed. The expressed HBsAg was determined by ELISA after infecting Bm N cells and pupae with recombinant virus BmPAK HBMp and BmPAK HBM (containing nonfusion HBV surface antigen middle protein gene), the results showed that the expression level of fusion HBsAg was increased by 60%～80% and the PreS2-Ag of fusion HBsAg was increased 8～10 times than nonfusion HBsAg. Kinetics of HBsAg and Pres2-Ag was different; the time of the highest expression level of Pres2-Ag was earlier one or two days than that of HBsAg. ELISA and Western blotting analyses showed that the expression of the HBsAg(PreS2+S) from the ATG of PreS2 is preferential because of the existence of polyhedrin gene sequence.