本文選題：偽狂犬病病毒變異株 + gE基因��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：豬偽狂犬病(Pseudorabies,PR)是由偽狂犬病病毒(Pseudorabies virus,PRV)引起的豬的一種以發(fā)熱、腦脊髓炎、呼吸系統(tǒng)和神經(jīng)系統(tǒng)障礙為主要特征的急性、熱性傳染病。免疫接種基因缺失株的弱毒疫苗是防控偽狂犬病的有效途徑之一。然而,自2011年以來,新的豬偽狂犬病疫情在中國北方的某些豬場(chǎng)最先爆發(fā),盡管這些豬場(chǎng)都免疫過了 Bartha-K61株等PRV疫苗,之后疫情逐步向全國大部分養(yǎng)豬場(chǎng)蔓延。研究表明此次疫情是由毒力明顯變強(qiáng)的PRV變異毒株引起的。有研究結(jié)果顯示PRV弱毒疫苗Bartha-K61雖然仍能提供一定的臨床保護(hù),但不能提供足夠的保護(hù)力以完全抵御此次流行的毒株,尤其對(duì)攻毒后的排毒保護(hù)效果很不理想。因此研制出一種針對(duì)PRV變異株的新型疫苗是我國養(yǎng)豬業(yè)的緊迫需求。本研究室從安徽省六安市某豬場(chǎng)的發(fā)病豬中分離了一株豬源偽狂犬病病毒株,該毒株能引起9周齡仔豬發(fā)病死亡,其毒力顯著增強(qiáng),命名為AH02LA株。進(jìn)一步以PRV AH02LA株基因組DNA為模板,分別擴(kuò)增出gE基因兩側(cè)的序列HI、H2作為同源重組臂,以綠色熒光蛋白(green fluorescent protein,GFP)為篩選標(biāo)記,構(gòu)建了GFP轉(zhuǎn)移載體質(zhì)粒pPRV-GFP(gE-)。采用磷酸鈣吸附法將pPRV-GFP(gE-)DNA與PRV-AH02LA病毒DNA共轉(zhuǎn)染原代CEF細(xì)胞,轉(zhuǎn)染24小時(shí)后在紫外光,(波長488nm)激發(fā)下觀察細(xì)胞病變,挑取發(fā)出綠色熒光的蝕斑,經(jīng)過3～4輪挑斑篩選和傳代,獲得純化的的重組病毒,命名為PRVAH02LA-GFP(gE-)。然后構(gòu)建質(zhì)粒pUC19-(PRV gE-)R,該質(zhì)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,同樣采用磷酸鈣吸附法將pUC19-(PRV gE-)R DNA與重組病毒PRV AH02LA-GFP(gE-)DNA共轉(zhuǎn)染原代CEF細(xì)胞,轉(zhuǎn)染24h后在紫外光(波長488nm)激發(fā)下觀察細(xì)胞病變,挑取不發(fā)綠色熒光的蝕斑,經(jīng)過2～3輪挑斑篩選和傳代,獲得純化的偽狂犬病病毒gE單基因缺失株,命名為偽狂犬病病毒LA-A株。對(duì)偽狂犬病病毒gE基因缺失株(LA-A株)的部分生物學(xué)特性進(jìn)行了鑒定。將PRV LA-A株分別感染CEF和BHK-21細(xì)胞,觀察病變情況并繪制PRV LA-A株在CEF和BHK-21細(xì)胞的生長動(dòng)力學(xué)曲線;用TCID50法檢測(cè)CEF和BHK-21細(xì)胞的病毒效價(jià),結(jié)果顯示,PRVLA-A株在CEF和BHK-21細(xì)胞中均能增殖,達(dá)到較高的病毒效價(jià),PRV LA-A株感染CEF細(xì)胞的病毒效價(jià)平均為106.5TCID50/mL,感染BHK-21細(xì)胞的病毒效價(jià)平均為107.8TCID50/mL。病毒的生長動(dòng)力學(xué)顯示,PRV LA-A株在CEF和BHK-21細(xì)胞上有相似的增殖周期,即在接毒一定時(shí)間內(nèi)增殖能力顯著上升,其后增殖平緩,甚至下降;但增殖效價(jià)有較大差異;從病毒的體外生長曲線可以看出,gE基因缺失病毒PRV LA-A株和親本病毒AH02LA株具有相似的生長動(dòng)力學(xué),在36h～48h都達(dá)到峰值,滴度最高可達(dá)109.5TCID50/mL以上,說明gE基因的缺失對(duì)病毒的復(fù)制幾乎沒有影響。安全性試驗(yàn)表明偽狂犬病病毒LA-A株對(duì)4周齡仔豬安全,且可以通過常規(guī)鑒別診斷方法區(qū)別偽狂犬病病毒LA-A株免疫豬只與自然感染豬只。對(duì)偽狂犬病病毒gE基因缺失株(LA-A株)滅活疫苗與偽狂犬活疫苗(Bartha-K61株)進(jìn)行免疫效力比較研究,免疫攻毒試驗(yàn)結(jié)果表明免疫Bartha K61株疫苗對(duì)免疫組豬僅能產(chǎn)生部分保護(hù),不能阻止發(fā)病和排毒。而接種LA-A株疫苗的所有豬只能產(chǎn)生對(duì)PRV野毒株AH02LA株的完全的臨床保護(hù),攻毒后排毒的時(shí)間大大縮短,強(qiáng)度顯著下降。表明PRV LA-A株作為疫苗株顯著優(yōu)于BarthaK61株。偽狂犬病病毒gE基因缺失株(LA-A株)滅活疫苗小鼠效力檢驗(yàn)表明PRV LA-A株對(duì)小鼠具有很好的免疫效力,對(duì)50LD50劑量的PRV AH02LA株能達(dá)到完全的保護(hù)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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