鸡源抗禽流感病毒Fab噬菌体抗体库的构建研究

【作者】 宫爱艳；

【导师】 蔡家利；

【作者基本信息】 西南农业大学 ， 临床兽医学， 2005， 硕士

【摘要】 禽流感（avian influenza）是由A型流感病毒引起的一种禽类感染或疾病综合症。禽流感病毒属正粘病毒科（Orthomyxoviridae）,为单股负链RNA病毒。甲型流感病毒除感染人外,还可感染猪、马、海洋哺乳动物和禽类。此病毒极易在禽鸟间散播,除可引起家禽大量死亡外,也可引起感染人的死亡。因此,禽流感病毒是一种危害禽类和人类健康的重要病原微生物。 对鸡源抗禽流感病毒抗体的研究不仅对疾病的预防和治疗有一定的作用,而且对进一步研究病毒在体内的致病机制提供了有利的工具。 我们首次运用噬菌体抗体库技术构建鸡源抗禽流感病毒Fab噬菌体抗体库。首先,我们从免疫鸡中提取外周血和脾脏,分离淋巴细胞,提取细胞总RNA,以其为模板,采用Oligo-dT引物逆转录合成cDNA,用一组鸡IgG Fab特异性引物,通过PCR方法扩增出一组抗体轻链基因和重链Fd片段基因。将抗体轻链基因克隆入pComb3噬菌粒载体,转化大肠杆菌XLI-Blue构建了库容量为2.5×10⁵的轻链基因抗体库,轻链基因的克隆效率约为50%。进一步将抗体重链Fd基因克隆入轻链抗体库中,转化大肠杆菌,构建了库容量为1×10⁵的鸡源抗禽流感病毒Fab抗体库。经鉴定,抗体轻、重链基因的克隆效率为40%。经辅助噬菌体感染,得到噬菌体滴度1×10¹³CFU/ml的鸡源噬菌体抗体库。用固相化AIVAg,对所构建的噬菌体抗体库进行了五轮的富集,第五轮洗脱下的噬菌体较第一轮增加了100倍,含有抗体轻链基因和重链Fd基因的克隆,也由富集前的15%增至65%。挑取几个阳性克隆进行测序,获得的序列与GeneBank相应的鸡IgG基因序列具有较高的同源性。 鸡源抗禽流感病毒Fab噬菌体抗体库的构建成功为抗禽流感病毒特异性抗体的筛选及构建鸡源抗禽流感病毒全抗体库打下了良好的基础,并为进一步研制用于禽流感治疗和抗禽流感病毒单抗制剂奠定了基础,同时也为研究禽流感病毒的体内致病机制提供了条件。更多还原

【Abstract】 Avian influenza (AI) is a disease infected by influenzavirus A. Avian influenza virus is the major etiologic agent of orthomyxoviridae. Influenzavirus A can infect swine, horse, mammal in ocean, poultry and human. Poultry and human which infected by this disease will also die.Phage display of antibody libraries has provided a powerful tool for the isolation of important viral pathogens .Large repertoires of antibodies can be displayed on the surface of filamentous phage particles, and antibodies with desired specificity can be isolated by panning on the antigen of interest.In this article ,we report the construction of chicken combinatorial immunoglobulin library to Avian Influenza Virus.The mRNA isolated from peripheral blood lymphocytes of chickens with AIV antibodies were reverse transcribed to the first strand cDNA using Oligo-dT as the primer.The genes of heavy Fd fragment and light chain of antibodies were amplified by a group of specific primers for chicken IgG Fab fragment. The gel purified light chain genes were firstly cloned into phagmid vector pComb3 to construct a chicken recombinatorial immunoglobulin library. The constructed library size was 1 x 10~5,and the cloning efficiencies of light chain genes were 50%.The heavy chain genes were subsequently combined with light chain genes randomly to constructed a combinatorial vectors.then transformed to E.Coli.XLI-Blue. the combinatorial Fabs libraries were constructed successfully. The chicken combinatorial Fab immunoglobulin library quantities were about 2.5x10~5 and the cloning efficiencies of Fab genes were 40%. Finally, the phage antibody library was further prepared by infecting chicken Fab combinatorial immunoglobulin library with help phage for displaying the titer of Fab fragment on the surface of phage .The titer of phages in library was lx10~13CFU/ml .The phage displaying antibody fragments were subjected to five rounds of paning with AIV antigen coated in solid phage. The eluted phages were enriched nearly 100-folder,and the percentase of recombinant clones increased from 15% to 65% after the five rounds of panning, choosed severy positive clones were sequenced and the data was analyzed by aligment with GeneBank, chicken immunoglobulin sequence was identified and confirmed. The light chains were identified as k type.We constructed chicken Fab recombinant library to AIV successfully. This specific Fab antibody combinatorial library laid a basis on panning and secreening specific Fab antibody to avian influenza virus. The antibodies panned and screened with puried AIV will be used as a tool to diagnose and study AIV. The antibodies will also be used as potential therapeutic medicine for the therapy of avian influenza after exposure to the virus.更多还原