Gene Cloning And Polycolonal Antibody Preparation And Tissue Expression Analysis Of Prolactin Receptor In The Goose

Prolactin receptor (PRLR) is a single transmembrane protein through which prolactin plays a wide variety of physiological roles in vertebrates. To understand its role in the goose, we cloned goose prolactin receptor gene (PRLR) cDNA with full length of 3,159bp by the method of rapid amplification of cDNA ends and prepared rabbit anti-goose PRLR polycolonal antibody, and then examined expression levels of mRNA and proteins in different organs of adult Hongze geese. At the same time, we checked and confirmed a SNP within a 5’-UTR of PRLR in Hongze geese.1. Cloning of goose prolactin receptor gene and tissue expression of the geneThe full length of cloned goose PRLR cDNA is 3,159bp long and it contains 443bp 5’ untranslated region,2,496bp coding sequence that presumably comprises at least 14 exons, and 220bp 3’untranslated region. The predicted goose PRLR contained 831 amino acids (aa) and exhibited identities of 87.7,85.2, and 84.8%with chicken, pigeon, and turkey PRLR, respectively. It comprised a signal peptide of 24aa at the N terminus, two ligand binding regions of the extracellular domain, each containing 2 pairs of cysteine residues and a pentapeptide of 5aa known as WS motif (Tpr-Ser-Glu-Tpr-Ser). Phylogenetic analysis revealed that the goose PRLR is highly conserved during evolution. Alignments of cloned cDNA fragments show that at least five PRLR transcripts expressed in testis of adult male goose. Two of them differ only by missing the last 95bp of exon 1, which is present in one transcript but absent in another; The third transcript is produced by another promoter with a distinct transcription initiation site from that for the above two transcripts, leading to creation of a novel exon 1 that is spliced to the third exon directly; Owing to lack of exon 4a, the fourth transcript can’t encode or may encode a truncated PRLR isoform; The fifth transcript encode a transcript that has only one ligand binding region which are shared by other vertebrate animal PRLRs except avian PRLRs. Reverse transcription PCR analyses show that the goose PRLR mRNA is widely expressed in testis, seminal duct, ovary, oviduct, kidney, large intestine, and small intestine and the highest abundance are presented in kidney, oviduct, large intestine and small intestine. 2. Preparation of rabbit anti-goose polycolonal antibody of goose prolactin receptor and tissue expression of the receptorAfter constructed exPRLR-pET-28a(+) was transformed into host bacteria E.coli Roster, a stable inducible prokaryotic expression system was established, and the protein exPRLR, whole extracellular domain of goose prolactin receptor, was induced to express by IPTG at 25℃. The expressed protein within supernatant and inclusion body of ultrasonic solution was purified by Ni-NTA HisBind Purification Kit, and rabbit anti-goose exPRLR polycolonal serum was then obtained after rabbits were immunized with purified and renatured exPRLR protein. The rabbit anti-goose exPRLR polycolonal antibody was purified with the method of ammonium sulfate precipitation from the serum and was then confirmed with the recombinant protein with extracellular domain of goose PRLR expressed by the established prokaryotic expression system and and expressed PRLR in goose tissues. At the same time, tissue expression of goose PRLR shows that there are three PRLR proteins existed in adult female goose tissues, and they may be the canonical PRLR protein and PRLR isoforms.3. A single nucleotide polymorphism in goose prolactin receptor gene and a novel DNA extraction solution for extracting genomic DNA extraction from very small number of animal cellsAlignments of cloned cDNA fragments exhibit two mismatches. The one located in the coding region is putatively regarded as a silent SNP, and it causes no change in protein. Another one located in 5’-UTR is confirmed to be a SNP within the core group of Hongze geese. In addition, in order to obtain genomic DNA from small number of goose feather follicle cells, we developed a novel DNA extraction solution, DNA MiniExtract, and its protocol. The solution allows us to extract genomic DNA from very small number of animal cells. The protocol is simple with only one step procedure and with low cost. The extracted genomic DNA is intact without any loss, thus insuring a sucessful DNA extraction without any repetition.