禽流感病毒血凝素基因的克隆及其DNA疫苗的免疫原性

Cloning of HA Gene of AIV and Immunogenicity of Its DNA Vaccine

禽流感病毒（ＡＩＶ）的表面结构蛋白血凝素（ＨＡ）是其主要保护性抗原。本研究参考已发表的Ｈ７亚型ＡＩＶ的ＨＡ基因序列，设计合成了１对Ｈ７ＨＡ特异引物，以ＡＩＶＡ／Ａｆｒｉ．Ｓｔａｒ．／Ｅｎｇ－Ｑ／９８３／７９／（Ｈ７Ｎ１）（Ａ／Ａｆｒｉ．Ｓｔａｒ．／Ｅｎｇ）核酸为模板，通过ＲＴ－ＰＣＲ扩增出１条１．７ｋｂｃＤＮＡ片段。将这一片段定向克隆到ｐＵＣ１８中，对其５′端及３′端部分序列测定后，确证其为ＨＡｃＤＮＡ。将ＨＡ基因置于ＳＶ４０启动子和增强子下游，构建了这一基因的真核表达质粒ｐＳＶＨ７。以此质粒１００μｇ肌肉注射免疫３周龄ＳＰＦ鸡６只，４周后以１００倍鸡胚感染剂量（ＥＩＤ）的ＨＡ基因同源病毒对所有鸡进行攻毒，１周后以棉拭子进行泄殖腔病毒分离；免疫后１～６周每周对所有鸡翅静脉采血，分离血清，检测ＨＩ抗体。结果，１００μｇ免疫组鸡病毒分离数为０／６，对照组为６／６；攻毒后１周免疫组鸡ＨＩ效价为１∶３２～１∶６４，对照组为１∶４～１∶１６。表明所构建的ＨＡ基因表达质粒可作为基因疫苗诱导鸡产生免疫保护反应。

One 1.7 kb DNA fragment was amplified by RTPCR from the RNA of AIV A/Afri. Star./ Eng, and proved to be HA gene by sequencing. The HA gene expression plasmid pSVH7 was constructed by putting the fragment under SV40 promoter and enhancer. 3weekold SPF chickens were inoculated with 100 μg DNA of plasmid pSVH7, 4 weeks later, all chickens were challenged with 100× egg infectious dose(EID) of AIV A/Afri. Star./ Eng, 1 week postchallenge all birds were sampled by cloacal swabbing to isolate virus. At every week postvaccination all birds were bled during the test period of 6 weeks for HI antibody detection. The virus isolation was negative in all vaccinated birds and positive in all control birds. Undetectable HI antibody were present in all birds prechallenged. The HI titers reached to 64 and 1 024 in vaccinated group, in contrast to 16 and 128 in control group at 1st week and 2nd week postchallenge respectively. These results showed that the plasmid pSVH7 designed as DNA vaccine would be able to elicit a firm protective immune response against AIV infection.