H5亚型禽流感病毒HA基因昆虫/杆状病毒系统的表达及对BALB/c小鼠的免疫保护

经RT-PCR扩增了禽流感病毒A/Goose/Guangdong/1／96 H5N1亚型1．7kb HA基因的cDNA,将其克隆到pMD18-T中并测序。亚克隆到杆状病毒转移载体pMelBacA的蜜蜂蜂毒素分泌信号下游中,测序正确后与线性化的杆状病毒DNA(Bac-N-BlueTM DNA)共转染Sf9昆虫细胞。将重组杆状病毒感染HFive细胞,72h左右收获细胞,超声波裂解,SDS—PAGE结果表明HA基因在重组杆状病毒感染的HFive细胞中获得表达。蛋白胶薄层扫描分析显示:表达的HA蛋白占重组杆状病毒感染细胞总蛋白含量的17．1％。Western-blot 及血凝实验结果显示,表达的禽流感H5N1亚型病毒HA蛋白具有生物学活性。表达的H5 HA蛋白定量乳化后,皮下多点注射免疫SPF 级BALB/c雌性小鼠,免疫后产生了H5 HA特异抗体,并在三免前后达到并保持较高水平。用致死剂量的HPAIV H5N1攻击小鼠,免疫组小鼠提供了100％的保护力,而对照组小鼠先后发病且死亡:为研制禽流感H5N1亚型病毒亚单位疫苗,防制禽流感奠定了基础。

Protection of Mice against a Lethal Avian H5 Subtype Influenza Challenge by Baculovirus-derived Hemagglutinin Vaccines

The HA gene of a highly pathogenic avian influenza virus (HPAIV) isolate (A/goose/Guangdong/l/96(H5Nl) was cloned and sequenced, then subcloned into the downstream of the baculovirus transfer vector pMelBacA. HA test and Western blot analysis demonstrated stable expression of the HA proteins. The oil-emulsion avian influenza subunit vaccine was prepared using expressed rH5HA (recombination H5HA protein) by allantoic fluid. SPF Balb/c mice were immunized three times with prepared subunit vaccine (contain 2.0 microg rH5HA per mice). Immunized mice generated a more consistent specific HI antibody response with an average geometric mean titer of 160 at 21 days of third post-vaccination. When challenged with highly pathogenic strains of the corresponding AI subtypes, the vaccinated mice were completely protected against lethal infection while all the negative control mice died, and no shedding at 5 days post infection. Vaccine protocols employing these recombinant HA proteins will not elicit an immune response against internal AI proteins and thus will not interfere with epidemiological surveys of natural influenza infections in the field. The results provide a system for the production of an effective recombinant influenza subunit vaccine that can easily be scaled up, and also establishes the baseline for developing people's avian influenza subunit vaccine.