表达IBDV VP2蛋白的重组火鸡疱疹病毒疫苗的构建及其免疫效力试验

发布时间：2019-01-19 10:03

【摘要】：传染性法氏囊病(Infectious bursal disease, IBD)是由传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)引起的雏鸡急性、高度接触性疾病。IBDV的感染对雏鸡的法氏囊造成损伤,从而导致免疫抑制,增加了机体对其他疾病的易感性。为了克服弱毒活疫苗和中等毒力活疫苗的缺陷,本研究通过在火鸡疱疹病毒(HVT) FC126毒株的US2复制非必需区,插入IBDV超强毒株的VP2基因,构建表达VP2蛋白的重组HVT疫苗毒株,旨在为研制预防IBD和马立克氏病(MD)的二联基因工程疫苗打下基础。本研究以IBDV XD2010超强毒株为模板,通过RT-PCR扩增其VP2基因,利用HVTFC126疫苗株基因组US2同源序列、CMV启动子和绿色荧光蛋白(EGFP)基因,构建含有VP2基因的转移载体质粒pCMV-EGFP-VP2,以磷酸钙沉淀法将转移载体质粒和HVTFC126基因组DNA共同转染鸡胚成纤维细胞(CEF),通过筛选、纯化得到带EGFP标志的重组病毒rHVT-EGFP-VP2。利用Cre重组酶去除rHVT-EGFP-VP2基因组中的EGFP基因,重新转染CEF细胞,经筛选、纯化得到不含EGFP基因的重组病毒rHVT-VP2。经PCR、间接免疫荧光(IFA)和western blot鉴定,重组病毒rHVT-VP2可正确表达VP2蛋白。在重组病毒rHVT-VP2对IBDV超强毒株的免疫保护试验中,设立rHVT-VP2疫苗组、中等毒力疫苗组(N87)、空白对照组和攻毒对照组,从各试验组免疫后的法氏囊重量／体重(囊体比)的变化、发病率、死亡率、攻毒后的增重、抗体水平等方面进行免疫保护效力的评价。结果发现,rHVT-VP2组免疫后法氏囊正常,而N87疫苗组免疫后法氏囊发生了萎缩；rHVT-VP2组及N87疫苗组攻毒后的发病率、死亡率与攻毒对照组相比差异极显著；攻毒后rHVT-VP2组体重增重显著高于N87疫苗组；利用本研究所建立的间接ELISA方法检测IBDV抗体,发现rHVT-VP2能够诱导机体产生较高水平的特异性抗体,并在一定时间内持续升高,抗体水平明显高于N87疫苗组；结合各组临床症状及器官病理变化,得出结论：重组病毒rHVT-VP2有效抵抗IBDV超强毒株的攻击,且免疫保护效力高于‘N87疫苗组。重组病毒rHVT-VP2对MDV的免疫保护试验中,rHVT-VP2组对MD保护率和HVTFC126疫苗组保护率相当,证明重组病毒rHVT-VP2能使机体产生较高的免疫保护力,足以抵抗MDV RB1B强毒株的攻击。综上所述,本试验所构建的重组病毒rHVT-VP2有效抵抗IBDV超强毒株攻击的同时,又能刺激机体对MDV的侵袭产生较高的免疫保护力,是理想的能够同时抵抗IBDV超强毒株及MDV攻击的新型疫苗。
[Abstract]:Infectious bursal disease (Infectious bursal disease, IBD) is an acute and highly contact disease in chicks caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV). The infection of IBDV causes damage to the bursa of Fabricius, which leads to immunosuppression. Increases the body's susceptibility to other diseases. In order to overcome the defects of live attenuated vaccine and moderately virulent live vaccine, the recombinant HVT vaccine strain expressing VP2 protein was constructed by inserting the VP2 gene of IBDV supervirulent strain into the non-essential region of US2 replication of turkey herpesvirus (HVT) FC126 strain. The aim is to lay a foundation for the development of dual genetic engineering vaccine for the prevention of IBD and Marek's disease (MD). In this study, the VP2 gene was amplified by RT-PCR using IBDV XD2010 supervirulent strain as a template. The genomic US2 homologous sequence of HVTFC126 vaccine strain, CMV promoter and green fluorescent protein (EGFP) gene were used. The transfer vector pCMV-EGFP-VP2, containing VP2 gene was constructed. The transfer vector plasmid and HVTFC126 genomic DNA were co-transfected into chicken embryo fibroblast (CEF), by calcium phosphate precipitation. Purification of Recombinant virus rHVT-EGFP-VP2. with EGFP Marker Cre recombinant enzyme was used to remove the EGFP gene from the rHVT-EGFP-VP2 genome, and then was retransfected into CEF cells. After screening, the recombinant virus rHVT-VP2. with no EGFP gene was purified. By PCR, indirect immunofluorescence (IFA) and western blot identification, the recombinant virus rHVT-VP2 can express VP2 protein correctly. In the immunoprotection test of recombinant virus rHVT-VP2 against IBDV supervirulent strain, rHVT-VP2 vaccine group, moderate virulence vaccine group (N87), blank control group and attack control group were established. The immune protection effectiveness was evaluated from the changes of bursa weight / body weight (thylakoid ratio), morbidity, mortality, weight gain after attack and antibody level. The results showed that the bursa of Fabricius was normal after immunization in rHVT-VP2 group, but shrank after immunization in N87 vaccine group, the morbidity and mortality of rHVT-VP2 group and N87 vaccine group were significantly different from those of control group. The weight gain of rHVT-VP2 group was significantly higher than that of N87 vaccine group. Using the indirect ELISA method established in this study to detect IBDV antibody, it was found that rHVT-VP2 could induce the body to produce a higher level of specific antibody, and the antibody level was significantly higher than that of N87 vaccine group. Combined with the clinical symptoms and pathological changes of organs in each group, it was concluded that the recombinant virus rHVT-VP2 could effectively resist the attack of IBDV super virulent strain, and the immune protection effect was higher than that of 'N87 vaccine group. In the immunoprotection test of recombinant virus rHVT-VP2 to MDV, the protective rate of rHVT-VP2 group to MD was similar to that of HVTFC126 vaccine group, which proved that the recombinant virus rHVT-VP2 could make the body produce higher immune protection ability, which could resist the attack of MDV RB1B virulent strain. In conclusion, the recombinant virus rHVT-VP2 constructed in this study can not only effectively resist the attack of IBDV super virulent strain, but also stimulate the body to produce higher immune protection against the invasion of MDV. It is an ideal new vaccine that can resist both IBDV super virulent strain and MDV attack.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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