【作者】 程宝艳；

【导师】 余为一；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 新城疫（ND）又称亚洲鸡瘟,是由新城疫病毒（NOV）引起的一种具有高度传染性的病毒性疾病。NDV病毒可引起鸡、火鸡、鸵鸟、鸽子、孔雀及其它野鸟等多种禽类发病。由于用于禽类的ND疫苗并不能完全阻止该病的发生,该病已造成巨大的经济损失。虽然我们对于造成免疫鸡群发生新城疫流行的根本原因尚不明确,但流行毒株的变异很可能是其中的重要因素之一。研究证明,在NDV感染过程中,位于病毒囊膜上的融合蛋白（F蛋白）是病毒穿入细胞,使宿主细胞产生融合和溶血的重要因子,同时也是病毒的主要保护性抗原。正因为如此,F蛋白已经成为研究新城疫基因工程疫苗和DNA疫苗等新型疫苗的首选抗原。为了探索NDV F蛋白抗原表位结构域基因在免疫应答中的作用及其机理,本研究运用分子克隆技术,对NDV F蛋白抗原表位结构域基因分别进行了克隆、表达和免疫原性鉴定。首先,根据GenBank中登录的NDV F48E9株F基因序列和载体pGEX-4T-1,pET-32-a的多克隆位点分别设计5对引物,应用反转录-聚合酶链式反应（RT-PCR）从接种NDV F48E9株的鸡胚尿囊液中扩增出相应大小的目的基因片段F509（509bp）、F₁（81bp）、F₂（108bp）、F₃（105bp）、F₄（102bp）。应用添加互补性酶切位点的基因工程方法,将抗原表位基因片段F₁、F₂、F₃拼接成多表位串联基因F306（306bp）。将片段F509插入载体pGEX-4T-1中,片段F₁、F₂、F₃、F₄、F306插入载体pET-32-a并转化宿主菌BL21,经双酶切鉴定重组质粒pGEX-4T-1/F509和pET-32-a/F306后,对F509和F306的核苷酸序列进行测定。结果表明本研究成功地克隆了NDV F蛋白抗原表位结构域基因及其多表位串联基因。其次,将上述构建成功的原核表达重组质粒pGEX-4T-1/F509、pET-32-a/F₁、pET-32-a/F₂、pET-32-a/F₃、pET-32-a/F₄和pET-32-a/F306转化大肠杆菌BL21,经异丙基-β-D-硫代半乳糖苷（IPTG）于37℃诱导培养4h,表达出含有谷胱甘肽-S-转移酶（GST）的融合蛋白GST-F509和含有组氨酸（His）的融合蛋白His-F₁、His-F₂His-F₃、His-F₄、His-F306;对表达条件进行相应的优化,确定了最佳诱导表达时间和诱导剂浓度;对表达蛋白的可溶性进行鉴定,结果表明表达的融合蛋白均以包涵体形式存在;应用优化的原核表达条件对含有质粒pGEX-4T-1/F509、pET-32-a/F₁、pET-32-a/F₂、pET-32-a/F₃、pET-32-a/F₄和pET-32-a/F306的BL21菌进行大量诱导表达,通过反复冻融和超声裂解诱导菌,获得浓度较高的GST-F509和His-F₁、His-F₂His-F₃、His-F₄、His-F306包涵体蛋白,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,提取了大量的可溶性GST-F509和His-F₁、His-F₂、His-F₃、His-F₄、His-F306融合蛋白。最后,通过Western-blot方法,用制备的鼠抗NDV阳性血清分别与获得的融合蛋白反应。结果表明含F蛋白抗原表位结构的融合蛋白GST-F509和His-F₁、His-F₂、His-F₃、His-F306具有良好的反应原性,而不含抗原表位的融合蛋白His-F₄则不能发生此特异性反应。利用上述纯化的GST-F509和His-F₁、His-F₂、His-F₃、His-F₄、His-F306融合蛋白作为抗原分别免疫小鼠制备鼠抗NDV F基因片段的多克隆抗体。琼脂扩散实验和ELISA实验结果表明制备的抗NDV F蛋白抗原表位结构域基因的多抗具有较高的效价。综上所述,本研究成功地克隆了NDV F蛋白抗原表位结构域基因及其多表位串联基因,构建了原核表达质粒,进行了原核蛋白表达和纯化,对融合蛋白的免疫原性进行初步的鉴定,并制备了效价高、反应性和特异性强的鼠源抗NDV F蛋白抗原表位结构域基因多克隆抗体,为进一步研究NDV F蛋白的表位疫苗奠定基础。更多还原

【Abstract】 Newcastle Disease（ND） which is also called chicken pest of Asia,caused by Newcastle disease virus（NDV）,is a kind of highly contagious disease.NDV can cause many kinds of poultry such as chickens,turkeys,ostriches,pigeons,peacocks and other wild birds ill.The vaccination against ND does not eradicate periodic outbreaks of the disease in immuunized poultry,and enormously make economic losses.Though the reason of the outbreaks of the disease in immunized poultry have not been known,the varation of NDV may play a major role.It is well known that F fusion protein located on the membrane of NDV plays an important role in the course of NDV infection,such as its penetration into host cells,fusion of viral and cellular membranes and the transference of viral genetic material in the cells.F protein is also a kind of viral protective antigen and crucially virulent structure.Therefore,it is considered as the most predominant antigen in the vaccine including genetic engineering vaccine and DNA vaccine.In order to research the function and mechanism of F protein in immune protective response,we cloned, expressed and identified the antigenity of epitope configuration region of Fgene.Firstly,according to F gene sequence of F48E9 registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1 and pET-32-a,five pairs of specific primers for the section fragment encoding chicken Newcastle disease virus F gene were designed and synthesized respectively.Using total RNA from allantoic of SPF chicken embryonating eggs,five fragment about 509bp,81bp,108bp,105bp,102bp were amplified by RT-PCR respectively.The tandem-arranged multiple epitope gene of the F gene was obtained by appending complementary endonudease of genetic engineering means.Then the genes were inserted into vector pGEX-4T-1 and pET-32-a respectively, and the recombinant plasmids were transformed into E.coli BL21.The recombinant plasmid pGEX-4T-1/F509was identified by EcoRI+SalI digestion and the recombinant plasmid pET-32-a /F306was identified by EcoRI+Xhol digestion.Then,the positive recombinant clone was sequenced and analyzed.The results suggest that the epitope configuration region of Fgene and the tandem-arranged multiple epitope gene of the F gene have been successfully cloned from allantoic of SPF chicken embryonating eggs.Secondly,the constructed recombinant plasmids pGEX-4T-1/ F509、pET-32-a/F₁、pET-32-a/F₂、pET-32-a/F₃、pET-32-a/F₄ and pET-32-a/F306 were transformed into E.coli BL21 and then induced to express GST-F509,His-F₁,His-F₂,His-F₃,His-F_a and His-F306 fusion protein by IPTG after cultivating for 4 hours.After optimizing prokaryotic expression conditions,4h was determined as the optimum induction time and 0.5mmol/L was determined as the optimum concentration of IPTG..The solubility of fusion proteins was identificated and the result indicated that expressed fusion proteins were located in inclusion bodies.Under the optimal condition,the recombinant plasmids were induced to express large-scale fusion proteins and the induced recombinant bacteria were lysed by freeze-thaw and sonication.The obtained GST-F509,His-F₁,His-F₂,His-F₃,His-F_a and His-F306 inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine,and the solubilized fusion proteins were obtained at last.Finally,the fusion proteins were reacted with the obtained antiserum against the NDV, and the results showed that GST-F509,His-F₁,His-F₂,His-F₃,and His-F306 fusion protein had strong specific reaction.Then mice were immunizated with the purified GST-F509,His-F₁, His-F₂,His-F₃,His-F_a and His-F306 fusion proteins and the polyclonal antiserum against the epitope configuration region of F gene were collected.A specific reaction appeared between the antibodies and GST-F509,His-F₁,His-F₂,His-F₃,and His-F306 fusion protein in agar diffusion assay and in ELISA,these results indicated that fusion proteins,GST-F509, His-F₁,His-F₂,His-F₃and His-F306 had strong antigenity.In conclusion,in this study the genes of epitope configuration region of Fgene were cloned successfully,the recombinant of plasmids pGEX-4T-1/F509、pET-32-a/F₁、pET-32-a/ F₂、pET-32-a/F₃、pET-32-a/F₄ and pET-32-a/F306 were constructed and the fusion proteins GST-F509,His-F₁,His-F₂ His-F₃,His-F_a and His-F306 were expressed in prokaryotic cells and purified.The antigenity of the fusion proteins were identified,and the specific polyclonal antiserum against the epitope configuration region of Fgene were prepared.All these would provide some experimental materials for the further studies on the epitope vaccine of NDV F gene.更多还原