Cloning,Polyclone Antibody Preparation And Expression Pattern Of Goat AP1M1 Gene

The protein of adaptor related protein complex 1 mu 1 subunit by the AP1M1 gene encoding.AP1M1 is one of the composite cohesion subunit protein AP-1 plays an important role in vesicle formation and coat protein sorting signal recognition,participate in exocytosis in secretory vesicle formation,cell membrane receptor recycling so the body a variety of important physiological functions.In recent years,studies have shown that AP1M1 may be involved in the development of testis,but the related research is less.In this study,the coding sequence of goat AP1M1 gene was successfully cloned from Dazu black goat,and the polyclonal antibody of goat AP1M1 molecule was prepared.Then,the expression of AP1M1 in various tissues and testis of adult Dazu black goat was detected.The results are as follows: 1.AP1M1 gene was cloned in goatSince there is no reported AP1M1 gene sequence in goat,by designing two pairs of specific primers,the sequence was cloned successfully by sequence splicing for the first time in goat(GenBank accession: MH155258).The total length of cDNA sequence was 1 661 bp.The open reading frame was 1 272 bp,encoding 423 amino acids,and predicted molecular weight was 48.59 KD.The sequence analysis showed that the gene was highly conserved in evolution.There are five protein-protein binding sites,no protein-nucleic acid binding sites,19 phosphorylation sites,which may play an important role in AP1M1 recognition of tyrosine based sorting signal "YXX?".It contains no transmembrane region,signal peptide structure,a Clat-adaptor domain and a Adap-comp domain.2.The polyclonal antibody of goat AP1M1 was prepared.In order to study the function of AP1M1,a prokaryotic expression vector pET30a(+)-his-AP1M1 was constructed.The results showed that the induction effect was better at 0.2 mM IPTG at 16 ? for 20 h,the recombinant protein existed mainly in the form of inclusion body,and the inclusion body was treated.The results showed that the inclusion body was washed with 4 M urea for 30 min and 8 M urea dissolved in 300 mm imidazolium nickel column,and the single target protein of SDS-PAGE band could be obtained,and the antiserum obtained after animal immunization was detected by Western blotting and indirect Elisa.The results showed that rabbit polyclonal antibody against goat AP1M1 was successfully obtained.3.Detect the 3.The expression patterns of AP1M1 in different tissues and testis of adult goats were analyzed.Q-PCR and Western blotting techniques were used to analyze the expression patterns of AP1M1 in different tissues and testicular development stages of adult goats.The results showed that the gene expression and protein expression patterns of AP1M1 in different tissues were the same,and they were found in goat heart,liver,spleen,lung,kidney,ovary,liver,spleen,lung,kidney and ovary.Testis,muscle are expressed,in which kidney expression is the highest,followed by testis.AP1M1 transcriptional expression in testis at different developmental stages showed that AP1M1 was expressed in testis from birth to maturation stage,and the expression level increased gradually with the development of testis,and the highest expression was observed at somatic maturity.This study provides a theoretical basis for the further study of the function of AP1M1 molecules,and also provides a basis for the study of the function of AP1M1 in testicular development.