重组真核表达载体pcDNA3.1(+)-ChIFN-α的构建及其与ND、IB二联活疫苗联用对鸡免疫功能的影响  

Construction of Recombinant Eukaryotic Expression Vector pcDNA3.1(+)-ChIFN-αand Its Effect on Chicken Immune Function in Combination with Live ND and IB Vaccine

作　　者：陈亚伟 杨文艳[1] 韩向新 宋艳红 吕小虎 陈书明[1] CHEN Yawei;YANG Wenyan;HAN Xiangxin;SONG Yanhong;LYU Xiaohu;CHEN Shuming(College of Veterinary Medicine,Shanxi Agricultural University,Shanxi Taigu 030801,China;Shanxi Provincial Animal Disease Control Center,Shanxi Taiyuan 030027,China;Shanxi Inspection and Testing Center(Institute of standard measurement technology),Shanxi Taiyuan 030027,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801 [2]山西省动物疫病预防控制中心,山西太原030027 [3]山西省检验检测中心(山西省标准计量技术研究院),山西太原030027

出　　处：《饲料工业》2022年第6期33-39,共7页Feed Industry

基　　金：山西省重点研发计划项目[201903D221013];吕梁市重点研发项目[2020NYGG4];山西省现代农业鸡产业技术体系专项[2021-2022];山西省优秀人才科技创新项目[201705D211029]。

摘　　要：为研究重组真核表达载体pcDNA3.1(+)-ChIFN-α与鸡新城疫(Newcastle disease,ND)、传染性支气管炎(Infectious bronchitis,IB)二联活疫苗联用最佳肌肉注射剂量及其对鸡免疫功能的影响。通过RT-PCR扩增鸡干扰素-α(IFN-α)基因,双酶切IFN-α基因和pcDNA3.1(+),T4 DNA ligase连接,构建重组真核表达载体pcDNA3.1(+)-ChIFN-α;挑选180只海兰褐公雏鸡随机分为6组,每组3个重复,每个重复10只。A组(生理盐水对照组)、B组(疫苗对照组)、C组[疫苗+100μg pcDNA3.1(+)对照组]、D组[疫苗+50μg pcDNA3.1(+)-ChIFN-α试验组]、E组[疫苗+100μg pcDNA3.1(+)-ChIFN-α试验组]、F组[疫苗+200μg pcDNA3.1(+)-ChIFN-α试验组];4日龄时,D、E、F组分别注射pcDNA3.1(+)-ChIFN-α50、100、200μg/只,C组注射pcDNA3.1(+)100μg/只,A、B组注射生理盐水300μL/只,7日龄时除A组外,其他各组均滴鼻、点眼免疫鸡ND、IB二联活疫苗,于49日龄时,对试验鸡进行肾型IB病毒(Nephropathogenic IB virus,NIBV)攻毒试验。PCR结果表明扩增出鸡IFN-α基因为582 bp,通过测序并与NCBI已发表的ChIFN-α基因(登录号EU367971)进行比对,同源性为100%;双酶切、菌液PCR及测序鉴定结果表明重组真核表达载体pcDNA3.1(+)-ChIFN-α构建成功;动物试验结果表明,不同剂量pcDNA3.1(+)-ChIFN-α均能提高鸡外周血阳性T、B淋巴细胞百分率;提高ND病毒(ND virus,NDV)、NIBV疫苗诱导的抗体水平,并能提高鸡对NIBV强毒株的免疫保护率,且100μg剂量组比疫苗对照组的免疫保护率提高10%。本研究确定重组真核表达载体pcDNA3.1(+)-ChIFN-α最佳注射剂量为100μg/只鸡,其不仅能发挥免疫佐剂作用,还可增强鸡ND、IB二联活疫苗免疫保护效果。The purpose of article was to study the optimal intramuscular injection dose of recombinant eu⁃karyotic expression vector pcDNA3.1(+)-ChIFN-αcombined with live Newcastle disease and infectious bronchitis vaccine and its effect on chicken immune function.ChIFN-αgene was amplified with RTPCR.The pcDNA3.1(+)-ChIFN-αwas construct⁃ed through the comection of T4 DNA ligase with the eukaryotic vector pcDNA3.1(+)and ChIFN-αdigested by double enzyme.One hundred and eighty 1-day-old Hy⁃Line brown rooster were ran⁃domly divided into six groups with three repli⁃cates per group and ten chickens per replicate.Group A(saline control group),group B(vaccine control group),group C[vaccine+100μg pcDNA3.1(+)control group],group D[vaccine+50μg pcD⁃NA3.1(+)-ChIFN-αexperimental group],group E[vaccine+100μg pcDNA3.1(+)-ChIFN-αexperimen⁃tal group],and group F[vaccine+200μg pcDNA3.1(+)-ChIFN-αexperimental group].At the age of four days,the chickens in group D,E and F were injected with pcDNA3.1(+)-ChIFN-α50μg,100μg and 200μg perchicken respectively,group C was injected with pcDNA3.1(+)100μg,perchicken and group A and B were injected with 300μL normal saline perchicken.At the age of seven days,all groups except group A were injected with live Newcastle disease and infectious bronchitis vaccine.At the age of 49 days chickens from all the groups were challenged with NIBV.The results of PCR showed that the amplified chicken IFN-αgene is 582 bp,and the homology was 100%by sequencing and comparing with the published ChIFN-αgene of NCBI(accession number EU367971).The results of double endonu⁃clease digestion,bacterial liquid PCR and sequencing showed that the recombinant eukaryotic expression vector pcDNA3.1(+)-ChIFN-αwas successfully constructed.The results of animal experiments showed that different doses of pcDNA3.1(+)-ChIFN-αcould increase the percentage of positive T and B lympho⁃cytes in chicken peripheral blood,increase the antibody level induced by Newcastle disease virus and ne⁃phropathogenic inf

关 键 词：ChIFN-α 真核表达载体 新城疫 传染性支气管炎 免疫佐剂 免疫功能 

分 类 号：S816.32[农业科学—饲料科学]