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目的选取新疆出血热病毒M基因(编码糖蛋白Gn,Gc)和S基因(编码核蛋白NP)部分基因片段,并进行部分片段的嵌合,分别插入至真核表达载体PVAX1中构建重组质粒,并将重组质粒分别免疫小鼠,评价其诱导的体液和细胞免疫应答的效果。方法将NP(aa1~482)、NP2(aa170~305)和糖蛋白Gn(aa529~820)、Gc(aa1050~1697)片段,及Gc-NP2片段分别构建到真核表达载体PVAX1上构建重组质粒,用重组质粒免疫小鼠,通过T细胞增殖试验,细胞因子含量测定和ELISA对免疫效果进行评价。结果经双酶切鉴定和DNA测序证实重组质粒构建正确。pVAX1-NP2实验组小鼠的血清抗体效价可以达到1∶6 400,pVAX1-NP2及pVAX1-Gc-NP2实验组小鼠脾脏T淋巴细胞增殖明显,两组的IFN-γ的表达水平也高达(865.15±6.29)pg/mL及(1727.21±33.93)pg/mL,与对照组相比,差异具有统计学意义(P<0.01)。pVAX1-NP2及pVAX1-Gn实验组IL-4的表达水平为(19.11±1.20)pg/mL和(20.07±1.67)pg/mL,都显著高于对照组的(9.35±1.82)pg/mL(P<0.05)。结论成功构建了多个重组真核表达载体,免疫小鼠后获得了效价高,特异性好的抗体。各重组质粒都激起了较强的体液免疫及细胞免疫,pVAX1-NP2及pVAX1-Gc-NP2实验组效果最好,可将其作为新疆出血热病毒的候选疫苗。
Objective To select partial gene fragments of M gene (encoding glycoprotein Gn, Gc) and S gene (encoding nucleoprotein NP) of Xinjiang hemorrhagic fever virus and to partially insert the gene fragment into the eukaryotic expression vector PVAX1 to construct a recombinant plasmid. The recombinant plasmids were immunized respectively to evaluate the effect of induced humoral and cellular immune responses. Methods The recombinant plasmids were constructed on the eukaryotic expression vector PVAX1, respectively. NP1 (aa1 ~ 482), NP2 (aa170 ~ 305) and glycoprotein Gn (aa529 ~ 820), Gc (aa1050 ~ 1697) , The mice were immunized with the recombinant plasmids, and the immune effects were evaluated by T cell proliferation assay, cytokine content assay and ELISA. Results Double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid was constructed correctly. The titer of serum antibody in pVAX1-NP2 experimental group mice reached 1: 400, the proliferation of T lymphocytes in the mice in pVAX1-NP2 and pVAX1-Gc-NP2 groups was significantly increased, and the expression level of IFN-γ in both groups was also up to (865.15 ± 6.29) pg / mL and (1727.21 ± 33.93) pg / mL, respectively. The difference was statistically significant (P <0.01) compared with the control group. The expression levels of IL-4 in pVAX1-NP2 and pVAX1-Gn experimental groups were (19.11 ± 1.20) pg / mL and (20.07 ± 1.67) pg / mL, which were significantly higher than those in the control group (9.35 ± 1.82 pg / P <0.05). Conclusion A number of recombinant eukaryotic expression vectors were constructed successfully. Antibodies with high titer and specificity were obtained after immunization. All recombinant plasmids provoked strong humoral and cellular immunity. The pVAX1-NP2 and pVAX1-Gc-NP2 experimental groups were the best, which could be used as vaccine candidates for Xinjiang hemorrhagic fever virus.