摘要
以本实验室分离保存的PRV SA强毒株，根据GenBank中伪狂犬病病毒全基因组序列设计了9对引物，扩增出目的基因，并将目的基因分别与T载体连接转化，挑取阳性菌落摇菌。分别进行PCR、单酶切和双酶切三种方法对阳性克隆进行了鉴定。成功克隆目的基因gI、gE、gD、gG、28k基因。对所得序列进行Blastn分析，并递交到DDBJ/EMBL/GenBank数据库中，登录号分别为AB440240（gG）、AB440243（gE）、AB440295（gI）和AB44024（Tk）。
针对gI、gE、28K、TK四个基因序列特异性位点，利用5条引物对强毒株SA株、gI-/gE-/PRV SA双基因缺失株和Bartha K61疫苗株进行了扩增，得到2061bp、1323bp、801bp和963bp特异性目的条带。Bartha K61疫苗株基因组检测最小浓度为136pg/μL。建立了针对野毒和两种疫苗株的PCR鉴别诊断方法。经测序得到gI-/gE-/PRV SA双基因缺失株gI-/gE-序列，GenBank登录号为GU262988.1，强毒株SA株TK基因GenBank登录号为AB44024。明确了gI-/gE-/PRV SA双基因缺失株（缺失gI和gE部分序列共计738bp）和PRV BarthaK61疫苗株(部分gI基因，全部的gE和US9基因和部分US2基因共计3489bp)确切位点。
建立了SYBR Green I实时定量PCR方法检测伪狂犬病毒gE基因缺失毒株。灵敏度比传统PCR方法的灵敏度高1000倍，前者可达1.75个拷贝/μL，后者为1.75×103个拷贝/μL。该方法可检测出PRV，不与PCV2、PPV、CSFV、PRRSV病毒发生交叉反应。利用gE基因建立的荧光定量方法对两株野毒株、我国疫苗市场中的6种伪狂犬病活疫苗进行了检测，结果符合病毒遗传背景。对山东区域分离伪狂犬阳性病料进行检测，发现2株gE基因阳性，测序分析，发现gE的抗原位点部分碱基突变。
建立了gE-ELISA检测方法的建立；检测由山东各地送检的不同猪场的240份疑似PRV感染血清样本，gE-PRV抗体检测阳性率27%，IDEXX HerdChekELISA Kit检测阳性率25.8％，阳性符合率95.19%。
建立了PRV gD-ELISA检测方法，确定了最佳反应条件和判定标准；检测了来自山东、河南、河北等地的306份血清样本，检出阳性258份，阴性58份，血清抗体阳性率为84.3%。
According to the whole genome sequence of pseudorabies virus (PRV) inGenBank, nine pairs of primers were designed to amplify the target gene of gI, gE, gD,gG,28K genes of PRV SA virulent strain. The gene inserted into T vector, positiveclones was identified by three methods of PCR, digested with single and doublerestriction enzyme respectively. The sequence Blastn analyzed and submitted to theDDBJ/EMBL/GenBank database. Respectively AB440240(gG), AB440243(gE),AB440295(gI) and AB44024(TK).
For four specific sites of gI, gE,28K, TK gene, five primers designed, SAvirulent strain, gI-/gE-/PRV SA mutant strain and Bartha K61strain was amplified,target band was2061bp,1323bp,801bp and963bp. Minimum detectableconcentration genome of Bartha K61vaccine strain was136pg/μL. Establishment testmethod of two vaccines and wild virus strain by PCR. Obtained mutant sequence bysequencing gI-/gE-/PRV SA strain, GenBank accession number GU262988.1.Clerified the exact mutant position of gI-/gE-/PRV SA strain (gI and gE deletionpartial sequences totaling738bp) and PRV Bartha K61vaccine strain (part gI gene,all of the gE and US9gene and part of the US2gene totaling3489bp).
Established a SYBR Green I real-time quantitative PCR to detect pseudorabiesvirus gE gene deletion strains. PCR was more sensitive1000times than the traditional,the former to1.75copies/μL, the later to1.75×103copies/μL. This method candetect PRV, no cross-reactive associated with PCV2, PPV, CSFV, PRRSV virus. InChina's vaccine market the six kinds of pseudorabies vaccine were tested, andfounded which complied viral genetic background. two positive gE gene PRV strainwere found in Shandong area from positive samples of PRV separating material.Some gE mutation of antigenic sites were founded after sequencing.
Established a gE-ELISA method for detection,240PRV sera samples of differentfarms were detected from from Shandong, PRV gE antibody positive rate of27%,IDEXX HerdChek ELISA Kit positive rate of25.8%, positive compliance rate 95.19%.Established a PRV gD-ELISA detection method to determine the optimalreaction conditions and criteria, detected from Shandong, Henan, Hebei and otherplaces306serum samples,258were detected positive, negative58copies, serumantibody positive rate84.3%.
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