马疱疹病毒I型gD基因真核重组质粒与原核表达蛋白联合免疫效果研究

发布时间：2018-09-12 08:31

【摘要】：马鼻肺炎(Equine rhinopneumonitis)被世界动物卫生组织(OIE)列为B类动物疫病,是由马疱疹病毒引起的一种急性、热性、高度接触性传染病。马疱疹病毒包括马疱疹病毒I型(Equine herpesvirus I)和马疱疹病毒IV型(Equine herpesvirus IV)。EHV-IV主要感染上呼吸道。EHV-I可影响多个器官系统,患畜表现为发热、食欲不振以及呼吸道等症状,有些可引起流产、新生马驹死亡、神经系统疾病等。虽然感染马鼻肺炎后死亡率不高,但是其引起的后遗症会给马匹带来巨大的健康隐患。根据Genbank上公布的马疱疹病毒I型(EHV-I)gD基因的序列分别设计一对特异性引物并进行扩增,获取的目的基因片段克隆到pMD19-T载体中,将测序鉴定正确的目的基因克隆到真核表达载体pVAX1上,PCR、酶切及DNA测序结果证实成功构建了含有马疱疹病毒I型gD基因的真核表达载体pVAX1-gD。通过介导重组质粒转染BHK-21细胞,经Western blot证实基因可在BHK-21细胞中表达。随后将gD重组质粒与gD蛋白分别与混合肌肉注射免疫小鼠,检测一免、二免的小鼠血清中和抗体效价,以及二免后的小鼠淋巴细胞增殖情况。ELISA与MTT检测结果表明,联合免疫能有效的诱导特异性血清中和抗体的产生,同时显著促进了特异性淋巴细胞的增殖。本研究成功构建了pVAX1-gD真核重组质粒,并且与pET-30a-gD蛋白联合免疫能诱导机体产生较高的体液免疫与细胞免疫,为马鼻肺炎基因工程疫苗的研制奠定了基础。
[Abstract]:Equine rhinopneumonia (Equine rhinopneumonitis) is an acute, feverish and highly contact infectious disease caused by equine herpesvirus by (OIE) of the World Health Organization (OIE). Equine herpesvirus includes equine herpesvirus type I (Equine herpesvirus I) and equine herpesvirus IV type (Equine herpesvirus IV). EHV-IV mainly infects the upper respiratory tract. EHV-I can affect many organ systems. The animals suffer from fever, loss of appetite and respiratory tract symptoms, some of which can cause miscarriage. The death of a newborn foal, neurological diseases, etc. Although the mortality rate is not high, the sequelae can bring great health risks to horses. According to the sequence of equine herpesvirus type I (EHV-I) gD gene published on Genbank, a pair of specific primers were designed and amplified. The obtained target gene fragment was cloned into pMD19-T vector. The correct target gene was cloned into the eukaryotic expression vector pVAX1. The results of restriction endonuclease digestion and DNA sequencing confirmed that the eukaryotic expression vector pVAX1-gD. containing the gD gene of equine herpesvirus type I was successfully constructed. The recombinant plasmid was transfected into BHK-21 cells, and Western blot confirmed that the gene could be expressed in BHK-21 cells. Then the gD recombinant plasmid and gD protein were injected into the mixed muscle to immunize mice respectively. The neutralization antibody titers of the first and second immunized mice were detected, and the lymphocyte proliferation of the mice after the two immunizations. Elisa and MTT showed that: 1. Combined immunity can effectively induce the production of specific serum neutralizing antibodies and promote the proliferation of specific lymphocytes. In this study, pVAX1-gD eukaryotic recombinant plasmid was successfully constructed and combined with pET-30a-gD protein to induce higher humoral and cellular immunity, which laid a foundation for the development of genetic engineering vaccine for equine rhinopneumonia.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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