Identification Of Anti-Mycobacterial Capacity Of HBD3and Production Anti-Mycobacteria Transgenic Cattle By Transfer The HBD3Gene

Mycobacterium tuberculosis of bovine (M.bovis) causes a chronic infectious disease inanimals, which is characterized by the formation of granulomas in the lung and other organs.This disease infects cattle and other domesticated animals as well as certain wildlifepopulations in numerous countries. M.bovis is spread worldwide and causes significanteconomic losses in animal production every year, especially in cattle production. Finding asuitable approach to reduce these losses in animal production has become significant andurgent. Previously studies have been shown that human β defensins (HBDs) participate in thecontrol of mycobacteria growth. Additionally, In the β-defensins family, HBD3have abroad-spectrum activity against both gram-negative and gram-positive bacteria atconcentrations much lower than those of other β-defensin family members. Thus, in this study,we firstly purified HBD3protein by affinity chromatography and examined its bioactivityagainst E.coli and staphylococcus and then tested its anti-mycobacteria capacity. Finally, weconstructed HBD3expression vector and obtained transgenic monoclonal cells by G418screening and then cultivated anti-mycobacterial transgenic cattle by SCNT technology.(1) This study firstly purified GST-HBD3protein by affinity chromatography, and thenobtained pure HBD3protein after digested by Factor Xa thrombase. Its bioactivity testshowed that purity HBD3protein could obviously inhibit multiplication of staphylococcuswhile has no obvious inhibition activity on E.coli.(2) To evaluate anti-mycobacterial capacity of HBD3, some tests has been operated,theresults showed that purified HBD3protein could inhibit mycobacteria multiplication atminimal inhibitory concentration20μg.ml-1. Additionally, HBD3protein was released inalveolar epithelial cells during mycobacteria invasion..(3) Distribution of muc1protein in cattle displayed that muc1promoter could appliedfor anti-mycobacterial research. Meanwhile, bioactivity analysis of Muc1promoter showedthat muc1promoter could drive GFP protein expression in alveolar epithelial cells. However,its bioactivity is weaker than CMV promoter in fundamental level. (4) Construction of immortalized cattle alveolar epithelial cells by transfer Htert gene.Interaction between the cell line and mycobacteria showed the immortalized cell line weestablished was a good target material for anti-mycobacterial research.(5) We constructed HBD3eukaryotic expression vector in this study.Anti-mycobacterial capacity were evaluated in alveolar epithelial cells. The results showedthat secretion of HBD3protein was obviously increased in cell medium during M.bovisinvasion(P<0.05). Anti-mycobacterial capacity exhibited that the excretive HBD3was able toinhibit mycobacteria multiplication. Additionally, mycobacteria susceptibility was obviouslyreduced in cattle transgenic epithelial cells than the normal one.(6) Embryonic development was evaluated by cleavage rate and blastocyst rate intransgenic embryo and non-transgenic one. There were no obviously differences between twotypes of embryos. Then we identified transgenic cattle after given birth. The PCR and otherexperiments exhibited that the cattle were contained the target gene. Secretion of HBD3protein in alveolar epithelial cells was obviously increased during M.bovis invasion (P<0.05),and its anti-mycobacterial capacity indicated the transgenic cattle could control mycobacterialmultiplication. Addtionally, mycobacteria susceptibility was obviously reduced in transgeniccattle epithelial cells than the normal one...