N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗的表达及免疫原性的影响

目的：研究N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗体外蛋白表达及小鼠体内体液免疫及细胞免疫应答的影响。方法：通过基因工程中定点突变技术，将乙型肝炎病毒表面抗原中蛋白（MHBs）中第4位氨基酸上连接的糖链去除，或将糖链依次移位至第5、6或7位氨基酸，来构建N-糖基化去除及移位的核酸疫苗，分别命名为Adr—dN4、Adr-N4—5、Adr—N4．6、Adr—N4．7。用上述核酸疫苗与野生型MHBs核酸疫苗（pSW3891／MHBs／Adr，简称Adr）及空载体质粒pSW3891分别用脂质体瞬时转染293T细胞，应用蛋白印迹法检测MHBs的表达。采用肌肉注射法，以各组疫苗分别对BALB／c小鼠于第0、2、4和6周进行免疫．用ELISA法检测小鼠血清中抗．HBs抗体、ELISPOT法检测小鼠表面抗原多肽特异性分泌IFN-T的脾细胞数量。结果：蛋白印迹法结果显示Adr、Adr-dN4、Adr—N4．5、Adr—N4—6、Adr-N4—7体外转染293T细胞后，均可以在293T细胞内表达，且Adr、Adr-N4—5、Adr-N4—7可将表达产物分泌到细胞外。ELISA及EISPOT结果表明：Adr免疫组小鼠抗-HBs终点滴度及表面抗原特异性分泌IFN-γ的脾细胞数量，均略高于其他免疫组小鼠，但与Adr—N4—5、Adr．N4—7相比无统计学差异（P〉0．05），与Adr-dN4和Adr—N4—6组相比有显著的统计学差异（P〈0．05）。结论：在第5或7位氨基酸附加N-连接糖链，能修补或替代Asn4连接糖链引导MHBs分泌的功能。HBs表达蛋白分泌到细胞外对诱导机体产生特异性细胞和体液免疫是至关重要的。

Effects of N-glycosylation Shift on Protein Expression in Vitro of Protein Nucleic Acid Vaccine and Immunogenicity in HBsAg

Objective： To investigate the expression and immunogenicity of DNA vaccine encoding middle hepatitis B surface antigen with relocating N-linked glycosylation. Methods：Site-directed mutagenesis were used to construct four DNA vaccines encoding middle hepatitis B surface antigen with relocating N-linked glycosylation named Adr-dN4, Adr-N4-5, Adr-N4-6 and Adr-N4-7, which transfer N-linked glycosylation sites from amino acid 4 to 5, 6 or 7 in preS2 domain. 293T cells were transiently transfected with Adr-dN4, Adr-N4-5, Adr-N4-6, Adr-N4-7, wild-type MHBs DNA vaccine （pSW3891/MHBs/Adr） and pSW3891 vector. The protein was measured by western blot. 7 weeks age BALB/c mice were used in the experiments. Each BALB/c mouse separately was intramuscularly injected with 100μg of each mutant or Adr or vector pSW3891 at 0, 2, 4 and 6 weeks. Anti-HBs in sera of mice were detected by ELISA. Peptide of HBsAg specific IFN-γ secreted splenocytes of mice were detected by ELISPOT. Results： There were the MHBs in the lysates of 293T cells transfected with each vaccine by western blot and in the supernatants in Adr, Adr-N4-5, Adr-N4-7. The peak Anti-HBs antibody titer and antigenic specific IFN-,y secreted splenocytes from immunized mice with Adr are slightly superior to with Adr-dN4, Adr-N4-5, Adr-N4-6, Adr-N4-7. But there was no significant difference between Adr and Adr-N4-5, Adr-N4-7 （P〉0.05）, while there was significant difference between Adr and Adr-dN4, Adr-N4-6 （P 〈0.05）. Conclusion： Defect by deleting N-linked glycosylation on amino acid 4 can be rescued by adding N-linked glycosylation on amino acid 5 or 7. Secreting of MHBs from the cells is essential to induce the specific cellular and humoral immunity.