摘要
【目的】制备针对奶山羊DDX3Y蛋白的特异性多克隆抗体,为奶山羊H-Y抗原蛋白DDX3Y的功能研究奠定基础。【方法】以奶山羊睾丸组织提取的RNA反转录所得的cDNA为模板,采用PCR方法扩增得到奶山羊DDX3Y基因CDs区全序列,测序后通过生物信息学方法比对分析确定DDX3Y蛋白N端1-120氨基酸及C端570-660氨基酸为抗原片段,然后用PCR分别克隆这2个片段的基因,最后利用搭桥PCR方法得到奶山羊DDX3Y截短抗原基因序列。将DDX3Y截短抗原基因序列连接pET32a(+)质粒,构建pET32a(+)-DDX3Y原核表达载体,将该载体导入到大肠杆菌BL21中诱导表达。通过Ni-NTA Resin纯化重组蛋白后免疫3月龄雌性新西兰大耳兔,采用间接ELISA(iELISA)法测定抗体效价,用Western blot测定抗体特异性。【结果】PCR扩增得到1 983 bp奶山羊DDX3Y基因CDs区全长序列及CDs区5′端360 bp(1-360 bp)序列和3′端273 bp(1 710-1 983 bp)序列。搭桥PCR获得了633 bp的DDX3Y截短抗原基因序列。成功构建了pET32a(+)-DDX3Y载体,并表达出分子质量约为42 ku的重组蛋白。制备出奶山羊DDX3Y截短抗原蛋白多克隆抗体,iELISA法检测抗体效价为1∶10~5;Western blot检测表明,制备的多克隆抗体能够特异性识别原核表达的奶山羊DDX3Y截短抗原蛋白以及公羊睾丸组织DDX3Y蛋白。【结论】成功制备出了奶山羊DDX3Y截短抗原蛋白多克隆抗体。
【Objective】This study prepared specific polyclonal antibody against DDX3Y protein of dairy goat to provide basis for studying functions of H-Y antigen protein DDX3Y in dairy goat.【Method】The DDX3Y gene CDs region full sequence of dairy goat was amplified by PCR using the cDNA obtained by reverse transcription of RNA from testis of dairy goat as template.After sequencing,the N-end 1-120 amino acids and C-end 570-660 amino acids of DDX3Y protein were identified as antigen fragments by bioinformatics comparative analysis.Then the genes of these two fragments were cloned by PCR,and the DDX3Y truncated antigen gene sequence of dairy goat was obtained by overlap PCR method.DDX3Y truncated antigen gene sequence was connected to the pET32a(+)plasmid to construct pET32a(+)-DDX3Y prokaryotic expression vector.The vector was introduced into E.coli BL21 to induce expression.The 3-month-old New Zealand White rabbits were immunized with the recombinant protein purified by Ni-NTA Resin.The antibody titer was determined by indirect ELISA(iELISA)method and the antibody specificity was determined by Western blot.【Result】PCR amplification obtained 1983 bp full-length sequence of DDX3Y gene CDs region,360 bp(1-360 bp)sequence of the CDs 5′end,and 273 bp(1710-1983 bp)sequence of the 3′end.The DDX3Y truncated antigen gene sequence of 633 bp was obtained by overlap PCR.The pET32a(+)-DDX3Y vector was successfully constructed and the recombinant protein with molecular weight of about 42 ku was expressed.The polyclonal antibody against DDX3Y truncated antigen protein was prepared,and iELISA assay antibody titer was 1∶105.Western blot analysis showed that the polyclonal antibody could specifically recognize DDX3Y truncated antigen protein produced by prokaryotic expression of dairy goat and DDX3Y protein in goat testis tissue.【Conclusion】Polyclonal antibody against DDX3Y truncated antigen protein was successfully prepared for goat.
作者
杜伟伟
代邦国
钟玉玲
史怀平
DU Weiwei;DAI Bangguo;ZHONG Yuling;SHI Huaiping(Key Laboratory of Animal Genetics,Breeding and Reproduction,College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2021年第1期1-9,16,共10页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31672398)
国家重点研发计划项目(2018YFD0501905)。
