伪狂犬病病毒gE基因缺失株（LA-A株）的构建及免疫效力研究

发布时间：2018-07-06 09:39

本文选题：伪狂犬病病毒变异株 + gE基因　；参考：《南京农业大学》2016年硕士论文

【摘要】：猪伪狂犬病(Pseudorabies,PR)是由伪狂犬病病毒(Pseudorabies virus,PRV)引起的猪的一种以发热、脑脊髓炎、呼吸系统和神经系统障碍为主要特征的急性、热性传染病。免疫接种基因缺失株的弱毒疫苗是防控伪狂犬病的有效途径之一。然而,自2011年以来,新的猪伪狂犬病疫情在中国北方的某些猪场最先爆发,尽管这些猪场都免疫过了 Bartha-K61株等PRV疫苗,之后疫情逐步向全国大部分养猪场蔓延。研究表明此次疫情是由毒力明显变强的PRV变异毒株引起的。有研究结果显示PRV弱毒疫苗Bartha-K61虽然仍能提供一定的临床保护,但不能提供足够的保护力以完全抵御此次流行的毒株,尤其对攻毒后的排毒保护效果很不理想。因此研制出一种针对PRV变异株的新型疫苗是我国养猪业的紧迫需求。本研究室从安徽省六安市某猪场的发病猪中分离了一株猪源伪狂犬病病毒株,该毒株能引起9周龄仔猪发病死亡,其毒力显著增强,命名为AH02LA株。进一步以PRV AH02LA株基因组DNA为模板,分别扩增出gE基因两侧的序列HI、H2作为同源重组臂,以绿色荧光蛋白(green fluorescent protein,GFP)为筛选标记,构建了GFP转移载体质粒pPRV-GFP(gE-)。采用磷酸钙吸附法将pPRV-GFP(gE-)DNA与PRV-AH02LA病毒DNA共转染原代CEF细胞,转染24小时后在紫外光,(波长488nm)激发下观察细胞病变,挑取发出绿色荧光的蚀斑,经过3～4轮挑斑筛选和传代,获得纯化的的重组病毒,命名为PRVAH02LA-GFP(gE-)。然后构建质粒pUC19-(PRV gE-)R,该质粒包含完整的gI基因和gE基因ORF的一部分(1299bp-1735bp)以及上下游序列,同样采用磷酸钙吸附法将pUC19-(PRV gE-)R DNA与重组病毒PRV AH02LA-GFP(gE-)DNA共转染原代CEF细胞,转染24h后在紫外光(波长488nm)激发下观察细胞病变,挑取不发绿色荧光的蚀斑,经过2～3轮挑斑筛选和传代,获得纯化的伪狂犬病病毒gE单基因缺失株,命名为伪狂犬病病毒LA-A株。对伪狂犬病病毒gE基因缺失株(LA-A株)的部分生物学特性进行了鉴定。将PRV LA-A株分别感染CEF和BHK-21细胞,观察病变情况并绘制PRV LA-A株在CEF和BHK-21细胞的生长动力学曲线;用TCID50法检测CEF和BHK-21细胞的病毒效价,结果显示,PRVLA-A株在CEF和BHK-21细胞中均能增殖,达到较高的病毒效价,PRV LA-A株感染CEF细胞的病毒效价平均为106.5TCID50/mL,感染BHK-21细胞的病毒效价平均为107.8TCID50/mL。病毒的生长动力学显示,PRV LA-A株在CEF和BHK-21细胞上有相似的增殖周期,即在接毒一定时间内增殖能力显著上升,其后增殖平缓,甚至下降;但增殖效价有较大差异;从病毒的体外生长曲线可以看出,gE基因缺失病毒PRV LA-A株和亲本病毒AH02LA株具有相似的生长动力学,在36h～48h都达到峰值,滴度最高可达109.5TCID50/mL以上,说明gE基因的缺失对病毒的复制几乎没有影响。安全性试验表明伪狂犬病病毒LA-A株对4周龄仔猪安全,且可以通过常规鉴别诊断方法区别伪狂犬病病毒LA-A株免疫猪只与自然感染猪只。对伪狂犬病病毒gE基因缺失株(LA-A株)灭活疫苗与伪狂犬活疫苗(Bartha-K61株)进行免疫效力比较研究,免疫攻毒试验结果表明免疫Bartha K61株疫苗对免疫组猪仅能产生部分保护,不能阻止发病和排毒。而接种LA-A株疫苗的所有猪只能产生对PRV野毒株AH02LA株的完全的临床保护,攻毒后排毒的时间大大缩短,强度显著下降。表明PRV LA-A株作为疫苗株显著优于BarthaK61株。伪狂犬病病毒gE基因缺失株(LA-A株)灭活疫苗小鼠效力检验表明PRV LA-A株对小鼠具有很好的免疫效力,对50LD50剂量的PRV AH02LA株能达到完全的保护。
[Abstract]:Pseudorabies (PR) is an acute, thermo infectious disease characterized by fever, encephalomyelitis, respiratory system and nervous system disorders, caused by Pseudorabies virus (PRV). The vaccine is one of the effective ways to prevent and control pseudorabies. However, from 2011, the vaccine is one of the effective ways to prevent and control Pseudorabies. The new swine pseudorabies outbreak has been the first outbreak in some pig farms in northern China. Although these pigs have been immune to PRV vaccines such as Bartha-K61 strain, the epidemic gradually spread to most pig farms throughout the country. The study showed that the epidemic was caused by the PRV mutant strain with strong virulence. The results showed that PRV was weak toxin. Although the vaccine Bartha-K61 still provides a certain clinical protection, it can not provide sufficient protection to fully resist the epidemic. Especially, the effect of detoxification after attack is not ideal. Therefore, developing a new vaccine against PRV variant is a pressing demand for the pig industry in China. This research room is from Lu'an, Anhui. A pig source pseudorabies virus strain was isolated in a pig farm. The strain could cause the death of 9 weeks old piglets, and the virulence was significantly enhanced. The AH02LA strain was named as the template of PRV AH02LA strain DNA. The sequence HI of the two sides of the gE gene was amplified, and H2 was used as the homologous recombination arm, and the green fluorescent protein (green fluores) was used. Cent protein, GFP) constructed the GFP transfer vector plasmid pPRV-GFP (gE-) for the screening marker. Using calcium phosphate adsorption method, pPRV-GFP (gE-) DNA and PRV-AH02LA virus DNA were co transfected to the primary CEF cells. After 24 hours transfection, the cell lesion was observed under ultraviolet light, and the green fluorescent plaque was selected and screened by 3~4 rounds of spot screening. The purified recombinant virus was named PRVAH02LA-GFP (gE-), and then the plasmid pUC19- (PRV gE-) R was constructed. The plasmid contained a complete gI gene and a part of the gE gene ORF (1299bp-1735bp) and the upstream and downstream sequences. The same calcium phosphate adsorption method was used to co transfect the pUC19- (PRV) and recombinant virus. The primary CEF cells were transfected with 24h under the excitation of ultraviolet light (wavelength 488nm) to observe the cell lesion and pick up the plaque without green fluorescence. After 2~3 rounds of screening and generation, the purified pseudorabies virus gE single gene deletion strain was named pseudorabies virus LA-A strain. The partial generation of the pseudorabies virus gE gene deletion strain (LA-A strain) was partially born. The physical characteristics were identified. CEF and BHK-21 cells were infected by PRV LA-A strains respectively. The pathological changes were observed and the growth kinetics of PRV LA-A strain in CEF and BHK-21 cells were plotted. The virus titer of CEF and BHK-21 cells was detected by TCID50 method. The results showed that the PRVLA-A strain could proliferate and achieve high viral titer. The virus titer of CEF cells infected with V LA-A was 106.5TCID50/mL, and the virus titer of infected BHK-21 cells was shown to be the growth kinetics of 107.8TCID50/mL. virus. The PRV LA-A strain had similar proliferation cycle on CEF and BHK-21 cells, that is, the proliferation energy increased significantly during the time of drug delivery, and the proliferation was slow and even decreased. The proliferation potency of gE gene deletion virus PRV LA-A strain and parent virus AH02LA strain have similar growth kinetics, and the peak value of 36h to 48h is up to 109.5TCID50/mL above, indicating that the loss of gE gene has almost no effect on the replication of the virus. The results showed that the pseudorabies virus LA-A strain was safe for 4 week old piglets and could differentiate the pseudorabies virus LA-A strains from the natural infected pigs by the conventional differential diagnosis method. The immune efficacy of the pseudorabies virus gE gene deletion (LA-A strain) inactivated vaccine and the pseudorabies live vaccine (Bartha-K61 strain) was compared and the immunization test was carried out. The results showed that the immunized Bartha K61 vaccine could only produce partial protection to the immune group, and could not prevent the disease and detoxification. All the pigs inoculated with LA-A vaccine could only produce complete clinical protection against the AH02LA strain of the PRV wild strain, and the time of detoxification was greatly shortened and the intensity decreased significantly. It showed that the PRV LA-A strain was used as a vaccine strain. The efficacy test of pseudorabies virus gE gene deletion strain (LA-A strain) inactivated vaccine mice showed that the PRV LA-A strain had good immune effect to mice, and the PRV AH02LA strain of 50LD50 dose could be fully protected.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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