山羊AP1M1基因表达分析与多克隆抗体制备
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摘要
为深入研究山羊AP1M1(adaptor related protein complex 1 subunit mu 1)在机体生长和繁殖中的作用,采用RT-PCR方法克隆了山羊AP1M1基因,并对其序列和编码蛋白进行分析;构建了pET-30α-AP1M1原核表达载体,免疫实验兔,制备AP1M1基因多克隆抗体;采用qPCR和Western blot检测比较AP1M1在山羊不同组织及睾丸不同发育时期的表达特征。结果显示,山羊AP1M1基因CDS为1 272 bp,编码423个氨基酸,具有1个Clat-adaptor结构域、1个Adap-comp结构域和5个蛋白质-蛋白质结合位点,在哺乳动物中极为保守。间接ELISA和Western blot结果表明,制备的兔抗pET-30α-AP1M1多克隆抗体效价为1∶128 000,可以与重组蛋白特异性结合。表达模式检测结果显示,AP1M1在成年山羊各组织中均有不同程度的表达,其中在肾脏的表达量最高,其次是睾丸,均极显著高于其他组织(P<0.01);此外,AP1M1在山羊不同发育时期睾丸组织中均有表达,但在性成熟时显著增加(P<0.01),体成熟时达到最高。本试验为进一步探索该基因在山羊睾丸中的功能奠定基础。
To explore the effect of AP1 M1(adaptor related protein complex 1 subunit mu 1)on goat growth and reproduction,AP1 M1 gene was cloned using RT-PCR.pET-30α-AP1 M1 prokaryotic expression vector was constructed and its polyclonal antibody was prepared.The expression levels of AP1 M1 in different goat tissues and in different stages of testicle were detected by qPCR and Western blot,respectively.The results showed that the CDS of AP1 M1 gene was 1 272 bp(encoding 423 amino acids)with a Clat-adaptor domain,an Adapt-comp domain and five protein-protein binding sites,which was highly conservative in mammals.The results of indirect ELISA and Western blot indicated that the rabbit anti-pET-30α-AP1 M1 polyclonal antibody were successfully generated with a titer of 1∶128 000 and can specifically bind to recombinant protein.The expression pattern analysis showed that AP1 M1 was expressed in various tissues of adult goat,and was highly expressed in kidney and testicular tissues(P<0.01).In addition,AP1 M1 was expressedin testicle throughout all stage of development,significantly increased at sexual maturity(P <0.01)and reached the highest at body maturation stage.This study lays the foundation for further exploration of the function of AP1 M1 gene in goat testicle.
To explore the effect of AP1 M1(adaptor related protein complex 1 subunit mu 1)on goat growth and reproduction,AP1 M1 gene was cloned using RT-PCR.pET-30α-AP1 M1 prokaryotic expression vector was constructed and its polyclonal antibody was prepared.The expression levels of AP1 M1 in different goat tissues and in different stages of testicle were detected by qPCR and Western blot,respectively.The results showed that the CDS of AP1 M1 gene was 1 272 bp(encoding 423 amino acids)with a Clat-adaptor domain,an Adapt-comp domain and five protein-protein binding sites,which was highly conservative in mammals.The results of indirect ELISA and Western blot indicated that the rabbit anti-pET-30α-AP1 M1 polyclonal antibody were successfully generated with a titer of 1∶128 000 and can specifically bind to recombinant protein.The expression pattern analysis showed that AP1 M1 was expressed in various tissues of adult goat,and was highly expressed in kidney and testicular tissues(P<0.01).In addition,AP1 M1 was expressedin testicle throughout all stage of development,significantly increased at sexual maturity(P <0.01)and reached the highest at body maturation stage.This study lays the foundation for further exploration of the function of AP1 M1 gene in goat testicle.
引文
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[13] NG F,TANG B L.Unconventional protein secretion in animal cells[J].Methods Mol Biol,2016,1459:31-46.
[14] MELLMAN I,NELSON W J.Coordinated protein sorting,targeting and distribution in polarized cells[J].Nat Rev Mol Cell Biol,2008,9(11):833-845.
[15] BONIFACINO J S.Adaptor proteins involved in polarized sorting[J].J Cell Biol,2014,204(1):7-17.
[16] SAWASDEE N,JUNKING M,NGAOJANLAR P,et al.Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 mu1A (AP-1 mu1A)[J].Biochem Biophys Res Commun,2010,401(1):85-91.
[17] DROBNIS E Z,NANGIA A K.Exogenous androgens and male reproduction[J].Adv Exp Med Biol,2017,1034:25-28.
[18] SU L,MRUK D D,LEE W M,et al.Differential effects of testosterone and TGF-beta3 on endocytic vesicle-mediated protein trafficking events at the blood-testis barrier[J].Exp Cell Res,2010,316(17):2945-2960.
[19] 任洁,陈福财,吴森,等.布尔山羊公羊身体质量与精液品质和生殖激素之间的关系[J].中国农学通报,2011(3):333-336.
[2] NAKATSU F,HASE K,OHNO H.The role of the clathrin adaptor AP-1:polarized sorting and beyond[J].Membranes (Basel),2014,4(4):747-763.
[3] FOLSCH H,PYPAERT M,SCHU P,et al.Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells[J].J Cell Biol,2001,152(3):595-606.
[4] MEDIGESHI G R,KRIKUNOVA M,RADHAK-RISHNAN K,et al.AP-1 membrane-cytoplasm recycling regulated by mu1A-adaptin[J].Traffic,2008,9(1):121-132.
[5] IIJIMA S,LEE Y J,ODE H,et al.A noncanonical mu-1A-binding motif in the N terminus of HIV-1 Nef determines its ability to downregulate major histocompatibility complex class Ⅰ in T lymphocytes[J].J Virol,2012,86(7):3944-3951.
[6] TAVARES L A,DA SILVAEM,DA SILVA-JANUARIOM E,et al.CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the gamma1 and gamma2 subunits of the AP-1 complex in protein trafficking[J].J Cell Sci,2017,130(2):429-443.
[7] DIB K,TIKHONOVA I G,IVETIC A,et al.The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit mu1A via a novel basic binding motif[J].J Biol Chem,2017,292(16):6703-6714.
[8] YONEYAMA Y,MATSUO M,TAKE K,et al.The AP-1 complex regulates intracellular localization of insulin receptor substrate 1,which is required for insulin-like growth factor I-dependent cell proliferation[J].Mol Cell Biol,2013,33(10):1991-2003.
[9] FRIOCOURT G,CHAFEY P,BILLUART P,et al.Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system[J].Mol Cell Neurosci,2001,18(3):307-319.
[10] GARIANO G,GUARIENTI M,BRESCIANI R,et al.Analysis of three mu1-AP1 subunits during zebrafish development[J].Dev Dyn,2014,243(2):299-314.
[11] OHNO H,TOMEMORI T,NAKATSU F,et al.Mu1B,a novel adaptor medium chain expressed in polarized epithelial cells[J].FEBS Lett,1999,449(2/3):215-220.
[12] GUO Y,YANG F,TANG X.An overview of protein secretion in yeast and animal cells[J].Methods Mol Biol,2017,1662:1-17.
[13] NG F,TANG B L.Unconventional protein secretion in animal cells[J].Methods Mol Biol,2016,1459:31-46.
[14] MELLMAN I,NELSON W J.Coordinated protein sorting,targeting and distribution in polarized cells[J].Nat Rev Mol Cell Biol,2008,9(11):833-845.
[15] BONIFACINO J S.Adaptor proteins involved in polarized sorting[J].J Cell Biol,2014,204(1):7-17.
[16] SAWASDEE N,JUNKING M,NGAOJANLAR P,et al.Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 mu1A (AP-1 mu1A)[J].Biochem Biophys Res Commun,2010,401(1):85-91.
[17] DROBNIS E Z,NANGIA A K.Exogenous androgens and male reproduction[J].Adv Exp Med Biol,2017,1034:25-28.
[18] SU L,MRUK D D,LEE W M,et al.Differential effects of testosterone and TGF-beta3 on endocytic vesicle-mediated protein trafficking events at the blood-testis barrier[J].Exp Cell Res,2010,316(17):2945-2960.
[19] 任洁,陈福财,吴森,等.布尔山羊公羊身体质量与精液品质和生殖激素之间的关系[J].中国农学通报,2011(3):333-336.