表达禽呼肠孤病毒σB、σC蛋白的重组马立克氏病毒的构建

发布时间：2018-11-14 20:41

【摘要】：禽呼肠孤病毒(Avian reovirus, ARV)感染可以引起的鸡和火鸡的以关节炎、键鞘炎、吸收障碍为特征的免疫抑制病。该病可导致鸡的死亡率增加、病鸡增重减少、饲料转化率下降、产蛋下降、孵化率/受精率降低,给养禽业带来巨大经济损失。国内外的研究表明目前使用的弱毒疫苗S1133仍可能具有一定的致病性,因此研制安全、有效的新型疫苗对ARV感染的防控具有重要意义。本研究采用RT-PCR扩增ARV GX/2010/1株的σB、σC基因,定向克隆至pEASY-M1表达载体中；再分别将σB、σC基因的表达盒与含有马立克氏病毒(MDV) CVI988疫苗毒株sorfl/sorf2序列pU/D载体相连,构建含σB、σC基因的转移载体质粒pU/D-aB和pU/D-aC。分别将pU/D-aB和pU/D-aC与含有GFP报告基因的MDV CVI988基因组DNA以磷酸钙沉淀法共转染CEF细胞,通过筛选、纯化获得重组病毒r-MDV-σB和r-MDV-aC。经PCR、Western blot和IFA分析,σB、σC基因分别正确插入到MDV CVI988毒株基因组中,并可以成功表达ARV σB蛋白和σC蛋白。本研究评价了1日龄SPF鸡接种重组病毒r-MDV-σB和r-MDV-aC后对ARV GX/2010/1株及MDV RB1B超强毒株的免疫保护效力。将70只1日龄SPF鸡随机分成5组：rMDV-σB免疫组、rMDV-aC免疫组、S1133疫苗免疫组、非免疫攻毒对照组和健康对照组,从发病率、死亡率、临床症状、病理组织学变化、对增重的影响和抗体水平等方面评价免疫效力。前两组的试验鸡1日龄时分别颈部皮下接种5000空斑形成单位(PFU)的r-MDV-aB和r-MDV-aC,第三组于7日龄接种1羽份的S1133疫苗；21日龄时,前四组试验鸡均用104.0EID50/鸡的ARV GX/2010/1毒株攻击,攻毒第14天进行剖杀。结果显示rMDV-σB免疫组和rMDV-σC免疫组在ARV攻毒后的发病率分别为40%和26.7%与非免疫攻毒对照组发病率100%存在显著差异(p0.05)；攻毒前后(21日龄～35日龄)各免疫组增重差异不显著(p0.05),但S1133疫苗组和rMDV-σB免疫组增重小于rMDV-σC免疫组。经临床检查,攻毒后rMDV-σC免疫组的脾脏病变较S1133疫苗免疫组和rMDV-σB免疫组轻微。本研究还评价了r-MDV-σB和r-MDV-σC分别对MDV RB1B超强毒株攻击的免疫保护效果。将80只1日龄SPF鸡随机分为4个试验组：rMDV-σB免疫组、rMDV-σC免疫组、CVI988/Rispens免疫组和攻毒对照组,每组20只试验鸡；rMDV-σB免疫组、rMDV-aC免疫组和CVI988/Rispens免疫组的试验鸡分别于1日龄时颈部皮下接种5000 PFU/鸡的相应疫苗；7日龄时,所有试验鸡经腹腔接种500 PFU/鸡的MDV RB1B超强毒株。连续观察90天,记录各试验组鸡的发病和死亡情况。rMDV-aB免疫组和rMDV-aC免疫组对MDVRBlB强毒株攻击能够提供完全保护,与MDV CVI988/Rispens免疫组的效果相当。综上所述,本研究构建的重组病毒rMDV-aC是较理想的新型疫苗候选毒株。
[Abstract]:Avian reovirus (Avian reovirus, ARV) infection may cause immunosuppressive diseases in chickens and turkeys characterized by arthritis, ketovitis, and dysabsorption. The disease could increase the mortality of chickens, decrease the weight gain, feed conversion rate, egg laying rate, hatching rate and fertilization rate, and bring great economic losses to the poultry industry. Studies at home and abroad show that the attenuated vaccine S1133 used at present may still have some pathogenicity, so it is of great significance to develop a safe and effective new vaccine for the prevention and control of ARV infection. In this study, the 蟽 B, 蟽 C genes of ARV GX/2010/1 strain were amplified by RT-PCR and cloned into pEASY-M1 expression vector. Then, the expression cassette of 蟽 B and 蟽 C gene was linked to the pU/D vector containing sorfl/sorf2 sequence of Marek's virus (MDV) CVI988 vaccine strain, respectively. The transfer vector pU/D-aB and pU/D-aC. containing 蟽 B and 蟽 C genes were constructed. The recombinant viruses r-MDV- 蟽 B and r-MDV-aCwere purified by co-transfection of pU/D-aB and pU/D-aC with MDV CVI988 genomic DNA containing GFP reporter gene by calcium phosphate precipitation. By PCR,Western blot and IFA analysis, 蟽 B and 蟽 C genes were inserted into the genome of MDV CVI988 strain correctly, and ARV 蟽 B protein and 蟽 C protein were successfully expressed. In this study, the immunological protective effects of 1 day old SPF chickens inoculated with recombinant virus r-MDV- 蟽 B and r-MDV-aC on ARV GX/2010/1 strain and MDV RB1B supervirulent strain were evaluated. Seventy 1-day-old SPF chickens were randomly divided into five groups: rMDV- 蟽 B immunized group, rMDV-aC immunized group, S1133 vaccine immunized group, non-immunized control group and healthy control group. The effect on weight gain and antibody level were evaluated. The first two groups were inoculated subcutaneously with r-MDV-aB and r-MDV-aCof 5000 plaque forming unit (PFU) in the neck of the first two groups, and the third group were inoculated with S1133 vaccine of 1 feather at 7 days of age. At the age of 21 days, the first four groups of chickens were all attacked with ARV GX/2010/1 strain of 104.0EID50/ chickens and killed on the 14th day of attack. The results showed that the incidence of rMDV- 蟽 B immunized group and rMDV- 蟽 C immunization group were 40% and 26.7% after ARV attack, respectively, compared with 100% of non-immunized control group (p0.05). There was no significant difference in weight gain between the two groups (p0.05), but the weight gain of S1133 vaccine group and rMDV- 蟽 B immunization group was lower than that of rMDV- 蟽 C immunization group. By clinical examination, the spleen lesion of rMDV- 蟽 C immunized group was less than that of S1133 vaccine and rMDV- 蟽 B immunization group. The immune protection of r-MDV- 蟽 B and r-MDV- 蟽 C against MDV RB1B strain was also evaluated. 80 1-day-old SPF chickens were randomly divided into four groups: rMDV- 蟽 B immunized group, rMDV- 蟽 C immunization group, CVI988/Rispens immunization group and control group. RMDV- 蟽 B immunized chickens, rMDV-aC immunized chickens and CVI988/Rispens immunized chickens were subcutaneously inoculated with 5000 PFU/ chickens at 1 day of age, and at 7 days old, all the tested chickens were intraperitoneally inoculated with MDV RB1B supervirulent strains of 500 PFU/ chickens. After 90 days of continuous observation, the incidence and death of chickens in each test group were recorded. RMDV-aB immunization group and rMDV-aC immunization group could provide complete protection against MDVRBlB virulent strain attack, which was similar to that of MDV CVI988/Rispens immunization group. In conclusion, the recombinant virus rMDV-aC constructed in this study is an ideal new vaccine candidate strain.
【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】：S852.65

【相似文献】