Study On Bacteriostatic Experiment In Vitro With Recombinant Protein Of Cattle Origin HMGN2

High mobility group chromosomal protein is the most abundant nonhistone family in nucleus of vertebrate and invertebrate.The members of this family are commonly existed in higher eukaryotes.It was named by the high electrophoretic mobility in the SDS-PAGE. HMG mainly binded to the field of nuclecome in the eukaryote activive transcriptional gene and built up the important function in maintaining the activive structure of chromosome,gene transcription and replication.HMG have three sub-families named HMGA,HMGB and HMGN.HMGN2,a member of HMGN,which located on the chromosome 1q35,composed of 90 amino acids with molecular weight 9.2kd.HMGN2 was isolated and purified from animals such as cow,mice and rabbit in 1980s.Its biological function was fully identified.Recent studies showed that HGMN2 had antimicrobial activity in addition to maintain the structure of chromosome,gene transcription and replication.The antibacterial mechanism of antimicrobial peptides was closely paid attention to and completely different from traditional antibiotics.In this study,we designed primers to amplify cattle origin HMGN2 gene from recombinant plasmid PUC57-HMGN2 and then constructed recombinant expression plasmid PET-32a(+)-HMGN2 and pGEX-KG-HMGN2.The prokaryotic expression of these two recombinant plasmids was induced by IPTG.The result showed that the expression of pGEX-KG-HMGN2 is higher than PET-32a(+)-HMGN2 at the same condition.The product of the high pGEX-KG-HMGN2recombinant expression plasmid existed in both supernatant and inclusion body.In the study,the way to extract and purify insoluble inclusion body of pGEX-KG-HMGN2 fusion protein was choosed.SDS-PAGE electrophoretic and western-blot analysis were used to measure their molecular weight and immunological activity of the fusion protein respectively.Staphylococcus aureus ATCC25923,pig Escherichia coli E052(O-9102),E126(O-138),Wh1(O-74) E107 (O-161),Wh3(O-139),X002(O-11),ETEC;chicken Escherichia coli O2,O78; Salmonella typhimurium,Streptococcus suis type 2 were choosed for bacteriostatic experiment by broth application and lamellate agarose diffusion assay.The results showed that the purified product of recombinant expression protein HMGN2 could inhibit the growth of E.coli Pig Wh3(O-139) and Staphylococcus aureus ATCC25923,MIC was 2.5μg/ml and 5μg/ml respectively and bacteriostasis was dose dependent according to the concentration.