| 刘浩1 邢益平1△ 贾一琼1 李军1 王世霞2 卢山2 黄祖瑚1.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[J].,2011,11(13):2443-2446 |
| 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 |
| Effects of N-glycosylation Shift on Protein Expression in Vitro of ProteinNucleic Acid Vaccine and Immunogenicity in HBsAg |
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| DOI： |
| 中文关键词: N- 糖基化 乙肝表面抗原中蛋白 免疫原性 核酸疫苗 |
| 英文关键词: N-linked glycosylation MHBs Immunogenicity DNA vaccine |
| 基金项目:江苏省兴卫工程重点学科基金（G:xk200715） |
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| 中文摘要: |
| 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-γ 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。 |
| 英文摘要: |
| 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-γ 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. |
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