摘要
传染性法氏囊病(infectious bursal disease，IBD)是由鸡传染性法氏囊病病毒(infectious bursal disease virus，IBDV)引起的幼龄鸡和火鸡的一种急性、高度接触性传染病。其主要危害是引起免疫抑制，导致鸡群对其它疫病的易感性增高和对疫苗的免疫应答降低。本研究的目的应用基因工程技术构建表达vvIBDV-VP2基因的重组鸡痘病毒并研究重组病毒的生物学特性，为IBDV重组疫苗的研制奠定基础。用甲醇毕赤酵母表达系统分泌表达vvIBDV-VP2蛋白并鉴定分析了其抗原性，为IBDV基因工程亚单位疫苗和诊断试剂的研制奠定基础。
研究主要从以下几个方面展开：
1．传染性法氏囊病病毒超强毒HN01株VP2基因的克隆和序列分析
提取vvIBDV感染的患病SPF鸡囊组织的总RNA为摸板，利用RT-RCR技术扩增出vvIBDV VP2基因，克隆入pMD-18T载体，构建含VP2基因的克隆载体pMD18T-VP2，通过对其酶切鉴定证明连入正确后测序。对测序结果进行了序列分析，发现测序结果和IBDV标准超强毒株UK661的核苷酸和氨基酸同源性分别为98.3％和99.8％。
2．表达vvIBDV-VP2基因重组鸡痘病毒的构建及重组病毒生物学特性的研究
利用PCR技术获得vvIBDV-VP2基因片段，并将其克隆入pEFgpt12S，成功构建pEFgpt-VP2重组鸡痘病毒转移载体。确定转染时亲本毒的用量及筛选重组病毒选择性培养基中霉酚酸的浓度后将鸡痘病毒转移载体pEFgpt-VP2与鸡痘病毒FPV 282E4株在次代CEF上进行同源重组。用霉酚酸连续筛选12代获得重组病毒rFPV-IBD-VP2。经间接免疫荧光试验(IFA)鉴定，可见阳性重组病毒表达产物产生特异性荧光，表明重组病毒成功表达了VP2基因且表达产物具有免疫原性；对亲本毒和重组毒进行TCID_(50)试验测定，两者的TCID_(50)值基本一致，表明外源基因重组入鸡痘病毒后不影响病毒的复制和生物学特性。将筛选到的重组鸡痘病毒rFPV-IBD-VP2继续在2％MEM中传5代，测定重组病毒分别在选择性培养基和普通培养基中的病毒滴度，并与亲本毒的滴度相比较，结果基本一致，说明重组病毒已得到纯化且比较稳定。
3．vvIBDV-VP2基因在毕赤酵母表达系统中的分泌性表达及抗原性鉴定用Primer5.0设计引物，以插入vvIBDV-VP2基因的pMD18-T为摸板，
中文摘要
利用PCR技术得到VPZ基因片段，并将vPZ基因片段亚克隆入分泌型毕赤
酵母表达载体pPICZ。一A中，成功构建重组表达质粒载体pPICz。一A一vPZ。
通过Zeocin的逐级筛选和提取酵母菌基因组进行PCR鉴定，筛选得到含VPZ
基因片段的高拷贝转化子18株，经甲醇诱导表达、表达条件优化、
SDS一PAGE，有12株转化子表达了VPZ基因。W己stem一biot试验和间接ELISA
结果表明表达产物都具有良好抗原性。
IBD is a acute contagious disease of little chicks and turkeys which caused by IBDV. It mainly leads to the immunosuppression of poultry, increases the susceptibility to other diseases and decreases the immune response to vaccine. This study aims to construct the recombinant poxvirus which can express the VP2 gene of vvIBDV and analysis its biology characterization, then provide the basis for the development of recombinant vaccines of IBDV. The VP2 protein of wIBDV was expressed with P.Pastoris expression system and its immunogenicity was identified. These provide the basis for the development of sub-unit vaccine and diagnosis reagent. The main contents are as follows:
1. Cloning and sequencing analysis for the VP2 gene of HN01 strain of very virulent infectious bursal disease Virus (vvIBDV)
Using total RNA of bursa of vvIBDV infectious SPF chicks as templet, the specific gene encoding IBDV VP2 was amplified by RT-PCR. The PCR product was successfully cloned into pMD-18T vector. The construct plasmid pMD-18T-VP2 was screened for the presence of MLUI and NHEI sites by restriction analysis, and the incorporated insert was further verified by dideoxy sequencing. Sequencing Analysis showed that the homology of target gene and animo acid with the standard wIBDV UK661 strain were 98.3% and 99.8% respectively.
2. Construction of recombinant fowlpox virus expressing vvIBDV-VP2 and its study of biological characteristic
The gene fragment encoding vvIBDV-VP2 was obtained by PCR. The PCR product was successfully cloned into pEFgpt!2S to construct recombinant fowlpox virus transfer plasmid vector pEFgpt-VP2. The quantity of parental FPV used in transfection and the optimal titer of Mycophenolic Acid (MPA) in selectable culture were determined. The recombinant fowlpox virus transfer plasmid vector pEFgpt-VP2 was cotransfected with fowlpox virus 282E4 strain at CEF by calcium phosphate-DNA coprecipitation method and the target linked
fragment was inserted into ORFl domain of FPV. Then the recombinant virus was selected with selectable culture containing MPA and was purified after 12 passages. Finally, Its specificity was identified positive on its infected CEF through Indirect Immunofluorescence Assay (IFA), this result shows that the recombinant fowlpox virus has successfully expressed the foreign genes and the tandem espressed products have antigenicity; Comparing the TCID50 of the recombinant fowlpox virus (rFPV-IBDV-VP2 ) with that of parental FPV, there was no obvious differences between them. The result shows that insertion of foreign genes into ORFl domain of FPV doesn't affect the propagation and biological characteristics of this virus.The selected recombinant fowlpox virus tandem espressing the wIBDV-VP2 was cultivated for 5 passages in normal culture (2% MEM) and then the liters of the recombinant in the selectable culture (2% MXHAT) and normal culture were tested and compared to the titer of parental FPV in normal culture. Th
ree values were same on the whole, which show that the recombinant was purified and its replication was stable relatively. 3. Secretive expression of wIBDV-VP2 in Pichia yeast and analysis of antigenicity
Using a pair of primers designed according to the relevant nucleotide sequence from GenBank, the specific gene encoding VP2 was amplified by PCR. The PCR product was inserted into the P. Pastoris secretory expression vector pPICZa-A. After the screen of zeocin and the identification of genomic of P. Pastoris, 18 recombinant strains with target gene were obtained. Under the optimal expression conditions, the target protein, which was proved by SDS-PAGE, was expressed in the supernatant of the twelve out of eighteen recombinant strains when induced with methanol. Western-blot and indirect ELISA analysis showed that the expression product had antigenicity.
引文
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