ABSTRACT
In order to study the biological function of pig BST-2 gene,the BST-2 gene was amplified with specific primers from porcine kidney tissue,and molecular characterization of BST-2 nuclectide and amino acid sequence were analyzed with bioinformatics tools and online server.Then the prokaryotic expression and tissue expression profile analysis was carried out.The results showed that the full length of pig BST-2 gene was 851 bp and contained 23 bp of 5'-UTR,294 bp of 3'-UTR and 534 bp of CDS and the gene encoded 177 aa.Amino acid sequence analysis of pig BST-2 protein showed 46.1％ identity with gorilla gorilla,41.7％ with cricetulus griseus,39.5％ with mus musculus,35.4％ with equus asinus,42.0％ with felis catus,40.5％ with bos mutus,44.4％ with macaca mulatta,38.7％ with ovis aries and 46.8％ with homo sapiens.BST-2 protein contained 2 transmembrane structure (27-49 aa and 154-176 aa),2 glycosylation sites and 14 potential phosphorylation sites including ATM,CK Ⅱ,PKA,PKC binding sites.The pig BST-2 protein was expressed in Vero cells after translated the recombinant plasmid FLAG-BST-2.Semiquantitative PCR results showed that BST-2 gene was expressed in all the tissues,especially in lymph nodes,thymus,tonsils,spleen,large intestine and small intestine.This study provide a foundation for further understanding the antiviral mechanism of pig BST-2 protein.
ABSTRACT
Objective To clone the human target gene HBVDNAPTP1 transactivated by hepatitis B virus DNA polymerase obtained by screening with suppression subtractive hybridization (SSH) and bioinformatics techniques. To construct prokaryotic expressive vector of HBVDNAPTP1 gene, induce the expression of recombinant protein in E. coli, and analyze the expression level of HBVDNAPTP1 gene in human tissues. Methods The DNA fragment of HBVDNAPTP1 was amplified by reverse transcription polymerase chain reaction (RT-PCR) taking mRNA from HepG2 cells as the template, and the correct DNA fragment was then inserted into inducible prokaryotic expressive vector pET-32a (+). The competent BL21 (DE3) E. coli was transformed, and then cultured and induced with IPTG. The expressed HBVDNAPTP1 was confirmed with Western blot. UniGene database was used to analyze the chromosome mapping and tissue expression profile of HBVDNAPTP1 gene. Results The DNA fragment of HBVDNAPTP1 was amplified by RT-PCR. HBVDNAPTP1 expressive vector was constructed. After transformation with pET-32a(+)-HBVDNAPTP1 and induction with IPTG, recombinant HBVDNAPTP1 was expressed and confirmed by Western blot. The expression of genomic location of HBVDNAPTP1 gene was low in multiple-tissues with the exception of pituitary gland, tonsil, tongue, thymus, trachea and umbilical cord. Conclusion The recombinant HBVDNAPTP1 gene could be expressed in prokaryotic expression system of E. coli. The chromosome mapping and tissue expression level of HBVDNAPTP1 gene is tentatively conceived.