The Primary Study On Expression Of Recombinant Human Beta Defensin-3 Gene In Mice Mammary Gland

Defensin is a kind of endogenous peptide, which restrain and kill Gram-positive bacteria, Gram-negative bacteria,fungus, virus and exert important effect in immunity system, with lower molecular weight, restrictive to heat, better water-dissolved, defensin has become distinctive value in research and development.Humanβ-defensin 3(hBD-3) found in 2001 is the third kind of humanβdefensin, it had obviously advantages on antibacterial activity compared with other defensins, which retraining against both Gram-positive bacteria and negative bacteria, Staphylococcus, Colon bacillus, Bacillus subtilis et al would be eradicated with several micrograms or milliliters of defensin. hBD-3 had expressed in E.Coli, but there was no activity without renaturation, Recombination protein produced by animal galactophore have more advantage over the traditional procaryon system with lower costs, larger output, higher biological activity and easier purified. Cow beta casein gene 5′control region and hBD-3 genome sequence were cloned and expression carrier pcDNA3.1(+) -hBD3 was constructed and injected into mice mammary gland Meantime, Recombinant carrier of cow beta casein gene 5′control region and hBD-3 also constructed to achieve especial expression in mice mammary gland. the result was as follows :①Cloning of human beta defensin-3 gene: According to the sequence of human beta defensin 3 on the GeneBank(NT077531), A pair of primers were designed and synthesized. The sequence consisting of 1392bp was obtained by PCR, Which was linked with pMD18-T.after identified with PCR and digested by enzyme. The sequencing shew the obtained DNA was homologous to NT077531 by 99.93% .②Cloning of bovine beta casein 5′control region : A pair of primers were designed and synthesized based on the gene sequence of bβ-CN on the GeneBank (X14711).The target DNA were obtained by PCR, Making up of 2334bp, Which was linked with pMD18-T.after identified with PCR and digested by enzyme. The sequencing indicted the target DNA was homogous to X14711 by 98.29% .③The Transient expression of human beta defensin-3 gene in mice mammary glands: Both the plasmids pMD18-hBD3 and pcDNA3.1(+) vector were digested with KpnI and XbaI. The fragments were purified and linked with T4 DNA Ligase and transferred into TG1. The recombinant plasmids were identified with PCR and digested with KpnI and XbaI , The results of sequencing revealed that the eukaryotic expression vector pcDNA3.1(+)-hBD3 consisting of human beta defensin-3 gene was successfully constructed .Then the recombinant expression plasmids were purified and were injected into lactating mice mammary glands with 25μg. Milk were collected after 24 hour and analysis of antimicrobial activity against E.coli showed that the milk did't show distinctive different with control group.