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目的构建H5N1高致病性人禽流感DNA疫苗表达质粒,并观察其免疫原性。方法分析H5N1人禽流感病毒HA基因的进化关系,人工合成人禽流感病毒HA基因(包含多数H5N1人禽流感病毒共有序列),构建含细胞毒性T淋巴细胞抗原4(CTLA4)和HA融合基因的真核表达质粒pVAX-CLHA及HA基因的真核表达质粒pVAX-HA,经酶切及测序鉴定正确后,转染293-T细胞,经RT-PCR法检测转染细胞中HA基因mRNA的转录水平,间接免疫荧光法(IFA)检测其表达;分别以质粒pVAX-CLHA及pVAX-HA免疫BALB/c小鼠,测定血清HI抗体效价。结果重组DNA疫苗表达质粒pVAX-CLHA和pVAX-HA经酶切及测序证明构建正确,转染的293-T细胞可检测到目的基因的转录及蛋白的表达;3次免疫后均能诱导BALB/c小鼠产生HI抗体,pVAX-CLHA诱导的HI抗体高于pVAX-HA。结论已成功构建了含H5N1HA及CTLA4融合基因的DNA疫苗表达质粒,其对小鼠具有良好的免疫效果,为H5N1高致病性人禽流感DNA疫苗的开发奠定了基础。
Objective To construct DNA vaccine expression plasmid for H5N1 HPAI and to observe its immunogenicity. Methods The evolutionary relationship of HA gene of H5N1 human avian influenza virus was analyzed. The HA gene of human avian influenza virus (including the consensus sequence of most H5N1 human avian influenza virus) was synthesized and the CTLA4 containing cytotoxic T lymphocyte antigen (CTLA4) and HA fusion gene Eukaryotic expression plasmid pVAX-CLHA and HA gene eukaryotic expression plasmid pVAX-HA, after digestion and sequencing identified correctly, transfected 293-T cells by RT-PCR detection of transfection of HA gene mRNA transcription BALB / c mice were immunized with plasmids pVAX-CLHA and pVAX-HA respectively, and the titer of serum HI antibody was measured. Results Recombinant DNA vaccine plasmid pVAX-CLHA and pVAX-HA were confirmed by restriction enzyme digestion and sequencing. The transfected 293-T cells could detect the transcription and expression of the target gene. After 3 immunizations, BALB / c mice produced HI antibodies, and pVAX-CLHA induced HI antibodies higher than pVAX-HA. Conclusion The DNA vaccine expression plasmid containing H5N1 HA and CTLA4 fusion gene has been successfully constructed and has good immunogenicity in mice, which lays the foundation for the development of H5N1 highly pathogenic human bird flu DNA vaccine.