Cloning Of Partial Sheep Gfra1and Preparing And Application Of Its Polyclone Antibody

Spermatagonial stem cells (SSCs) and its product protein antibody play a pivotal role in the spermatagonial stem cell research. cDNA sequences of surface marker gene can be used for identifying of the transcriptional expression of the SSCs. protein production of surface marker gene can be used for separation and purification of SSCs by Flow Cytometer, or for the identifying of SSCs by immunofluorescence staining. So far, the long-term purified and cultured large animal SSCs have not been reported successful, which has the relationship with the lack of SSCs surface marker DNA sequences and antibodies. GFRal is the receptor proteins of GDNF on the cell membrane of SSCs and is one of the surface marker genes of spermatagonial stem cells (SSCs). GFRal also plays an important role in the regulation of SSCs proliferation and differentiation. GDNF bind with RET protein through the GFRal receptor of SSCs membrane surface, causing RET dimerization and phosphorylation of tyrosine residual base site and starting signaling pathways. So far, the sheep Gfral gene sequences has not been reported which impact of the sheep SSCs researching. So it is necessary to research sheep Gfral gene for establishing the foundation of spermatogenesis and separating SSCs.1. Cloning of sheep Gfral geneA pair of primers for amplifying the conserved fragment of the Gfral gene was designed according to the homo sapiens, Rattus norvegicus Mus musculus, Bos Taurus and Pongo abelii Gfral gene from GeneBank. With sheep testicular tissue cDNA as templates, sheep Gfral middle sequence of1041bp was obtained by RT-PCR amplification. The PCR products were cloned into the pMD-19T vector for sequencing. Another pair of primers was designed for cloning forepart sequence of Gfrα1. Gfrα1sequences of forepart sequence and middle sequence were joined together. The cloned partial Gfral gene was1312bp in length, including initiator codon and an ORF of131lbp which encoded437amino acid. The nucleotide sequence of sheep Gfral gene was compared with the counterpart sequences of Bos Taurus, homo sapiens, Pongo abelii, Rattus norvegicus and Mus musculus. The nucleotide homology was separately98.5%,91.3%,91.0%,88.9%and88.0%. The result demonstrated that Gfra1gene sequences are highly conserved in the evolutionary process. Sheep as a ruminant has this species specific in Gfra1gene sequences.2. Construction of prokaryotic expression vector for preparation and purification of epitope polypeptide of GFRalAccording to the cloned Gfrα1gene sequence, we forecast its epitope and then insert cDNA into pET-44a(+)expression vector. Subsequently, the recombinant was induced by IPTG. The recombinant epitope polypeptide was purified according to Affinity column chromatography. Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate18.2KD. Expression of GFRal epitope polypeptide in BL21(DE3) and purification of epitope polypeptide lay a good foundation for preparation of polyclonal antibody.3. Production of polyclonal antibody by means of injection of S180ascites tumor cells and Identification of polyclonal antibodyThe reeombinant his-GFRα1epitope polypeptide purified by affinity chromatography was used as antigen to immunize6to8weeks of C57mice. It was needed for immunizing five times with an interval of12days.After a week of the last immune, human fibrosarcoma cell(S180)was inoculated into C57mice in order to obtain the high level antibody. Until the mice abdomen has increased, ascites containing his-GFRal polyclonal antibodies was collected. The specifity of his-GFRα1epitope polypeptide was tested by indirect ELISA and the titer was1:800showing the higher specificity. Western blotting of Sheep testicular tissue GFRα1protein showed specific band. The molecular weight of specific band was about50KD which was consistent with the size of the predicted GFRα1protein. So it was proved that obtained polyclonal antibodies of this study has high specificity.4. Identification of vitro cultured sheep SSCs by sheep GFRα1polyclonal antibodyLong-term cultured sheep SSCs were identified by GFRα1and PLZF immunol fluorence staining. It has been proved that PLZF is a marker of sheep SSCs. The result of Immunol fluorence staining of long-term cultured sheep SSCs with PLZF polyclonal antibody showed positive and trophoblast cells negative. It is means that our lab has obtained long-term cultured sheep SSCs. Immunol fluorence staining.showed that GFRα1positive cells overlapped with PLZF positive cells. These results illustrated that the laboratory-made GFRα1antibodies can be used as a marker of sheep SSCs which also can be used for identification and analysis for the sheep SSCs. The results of this study provide a method for the research and operation of the sheep SSCs, and lay the foundation for the research of morphology and distribution of sheep SSCs and for genetic study and transgenic technology of SSCs.