猪催乳素的真核表达与生物活性验证  

Eukaryotic expression and bioactivity verification of porcine prolactin

作　　者：谢社风 韩贝贝 高凤磊[2] 马莹 李莉[1] 张守全[1] 邹娴 卫恒习[1,4] XIE Shefeng;HAN Beibei;GAO Fenglei;MA Ying;LI Li;ZHANG Shouquan;ZOU Xian;WEI Hengxi(National Engineering Research Center for Breeding Swine Industry/Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/College of Animal Science,South China Agricultural University,Guangzhou 510642,China;School of Tropical Agriculture and Forestry,College of Guangdong Agriculture Industry Business Polytechnic,Guangzhou 510507,China;Institute of Animal Science,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Maoming Branch,Guangdong Laboratory for Lingnan Modern Agriculture,Maoming 525000,China)

机构地区：[1]国家生猪种业工程技术研究中心/广东省农业动物基因组学与分子育种重点实验室/华南农业大学动物科学学院,广东广州510642 [2]广东农工商职业技术学院热带农林学院,广东广州510507 [3]广东省农业科学院动物科学研究所,广东广州510640 [4]岭南现代农业科学与技术广东省实验室茂名分中心,广东茂名525000

出　　处：《华南农业大学学报》2024年第2期179-189,共11页Journal of South China Agricultural University

基　　金：广东省重点领域研发计划(2022B0202110002);广东省自然科学基金(2020A1515010976);广东省“珠江人才计划”本土创新科研团队项目(2019BT02N630)。

摘　　要：【目的】催乳素(Prolactin,PRL)具有广泛的生理调节作用,但其多效性机制仍不清楚。为了更好地研究猪PRL的多效性,本研究制备猪源PRL真核重组蛋白并验证其生物活性。【方法】利用分子克隆技术将猪PRL基因克隆到慢病毒表达载体pCDH-CMV-MCS-EF1-GFP+Puro中,经慢病毒包装获得携带猪PRL基因的PRL-慢病毒;用浓缩的PRL-慢病毒感染CHO-K1细胞,经嘌呤霉素筛选后,获得能够分泌PRL重组蛋白的阳性细胞系CHO-K1-PRL;利用镍柱亲和层析法对重组蛋白进行纯化并进行LC-MS/MS质谱鉴定,利用HC11细胞体外培养体系验证PRL重组蛋白的生物活性。【结果】成功构建了携带猪PRL基因的pCDH-CMV-6His-PRL-6HisEF1-GFP+Puro慢病毒表达载体;包装及浓缩后的PRL-慢病毒滴度为9.9×10^(8) TU/mL,其感染的CHO-K1细胞经嘌呤霉素筛选后得到阳性细胞系CHO-K1-PRL;从CHO-K1-PRL细胞培养液中成功纯化出重组蛋白,质量浓度为50μg/mL,LC-MS/MS质谱分析的覆盖率达94%,鉴定为猪PRL重组蛋白;重组PRL具有促进HC11细胞增殖及酪蛋白表达的生物活性。【结论】构建的细胞系CHO-K1-PRL可稳定表达具有生物活性的猪重组PRL,为猪PRL功能的研究和生产应用奠定了基础。【Objective】Prolactin(PRL)has a wide range of physiological regulatory effects,but its pleiotropic mechanism is still unclear.In order to investigate the pleiotropy of porcine PRL,we obtain porcine PRL eukaryotic recombinant protein and verify its biological activity.【Method】The porcine PRL gene was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-GFP+Puro by molecular cloning technology,and the PRL-lentivirus carrying porcine PRL gene was obtained by lentivirus packaging.CHO-K1 cells were infected by the concentrated PRL-lentivirus solution,and the positive cell line named CHO-K1-PRL,which could secrete recombinant PRL protein,was obtained after purinomycin screening.The recombinant protein was purified by nickel column affinity chromatography,and identified by LC-MS/MS mass spectrometry.The biological activity of recombinant PRL was verified by adding recombinant PRL into HC11 cell culture system in vitro.【Result】The recombinant expression vector pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro carrying porcine PRL gene was successfully constructed.The titer of PRL-lentivirus after concentrating was 9.9×10^(8) TU/mL,and the positive cell line CHO-K1-PRL was obtained after puromycin screening.The recombinant protein with the mass concentration of 50μg/mL was successfully purified from the supernatant of CHO-K1-PRL cells.The recombinant porcine PRL protein was identified as porcine PRL by LC-MS/MS mass spectrometry,and the coverage rate of recombinant PRL was 94%.The recombinant PRL had the biological activity of promoting the proliferation and casein expression of HC11 cells.【Conclusion】The cell line CHO-K1-PRL constructed in this study can stably express porcine recombinant PRL with biological activity,which lays a foundation for the functional research,production and application of porcine PRL.

关 键 词：猪 催乳素 CHO-K1细胞 真核表达 慢病毒载体 重组蛋白 

分 类 号：S828.2[农业科学—畜牧学]