摘要
为了制备奶山羊乳铁蛋白的多克隆抗体,为进一步的试验提供材料,该研究运用软件分析并选取最适合原核表达的LF序列进行扩增,将扩增出的序列与pET-32a(+)质粒连接,形成原核表达质粒pET-32a-LF,将质粒转化入大肠杆菌,在最适条件(37℃、0.5 mmol/L IPTG、4 h)下诱导表达出大量蛋白。通过Ni-NTA Resin法将蛋白纯化后皮下注射4月龄新西兰大耳兔,免疫注射结束后采集抗体血清,并用间接酶联免疫吸附法和免疫印迹法检测多抗的效价及特异性。结果显示克隆得到适合原核表达的279 bp的LF CDS片段,pET-32a(+)-LF原核表达载体成功构建并顺利表达(未形成包涵体),表达的截短蛋白大小为10 kD。抗体效价达到1:10^(7);免疫印迹试验结果显示,原核表达的LF蛋白和山羊乳腺上皮细胞中的LF蛋白能够被该抗体特异性识别。
To prepare lactoferrin polyclonal antibodies of dairy goats and provide materials for further experiments,this study used software to analyze and select the most suitable LF sequence for prokaryotic expression and amplify it.The amplified sequence and pET-32a(+)plasmid was connected to form the prokaryotic expression plasmid pET-32a-LF,the plasmid was then transformed into E.coli,and a large amount of protein was induced to be expressed under optimal conditions(37℃,0.5mmol/L IPTG,4 h).The protein was purified by Ni-NTA Resin method and injected subcutaneously into 4-month-old New Zealand rabbits.After the immunization injection,antibody serum was collected,and the titer and specificity of the prepared polyclonal antibody were detected by indirect enzyme-linked immunosorbent assay and immunoblotting.The results showed that a 279 bp LF CDS fragment suitable for prokaryotic expression was cloned.The pET-32a(+)-LF prokaryotic expression vector was successfully constructed and successfully expressed(without inclusion bodies).The size of the truncated protein expressed was 10 kDa.Antibody titer reached 1:10^(7);immunoblot test results showed that LF protein expressed in prokaryotic cells and LF protein in goat mammary epithelial cells can be specifically recognized by the antibody.
作者
钟玉玲
代邦国
杜伟伟
张华文
李纯
史怀平
ZHONG Yuling;DAI Bangguo;DU Weiwei;ZHANG Huawen;LI Chun;SHI Huaiping(College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,China)
出处
《家畜生态学报》
北大核心
2022年第2期13-19,共7页
Journal of Domestic Animal Ecology
基金
陕西省创新能力支撑计划(2021TD-31)
西安市科技计划项目(20NYYF0030)
陕西省农业科技创新驱动项目(NYKJ-2021-YL(XN)09)。
