The Effective Expression Of The CecropinB In Goat Mammary Gland Epithelial Cell Line

This study was designed to construct the eukaryotic expression vector by gene recombination technology, and express cecropinB gene in the goat mammary epithelial cells. It aimed at the expression of cecropinB gene at a high level in the mammary glands of goats,and provided a viable technical means and credible theories on production of transgenic animals and transgenic milk, improvement of milk quality for the future. In addition, the eukaryotic expression vector constructed in this study contained the whole EGFP gene sequence, and its downstream regulatory sequence ended with restriction endonuclease sites,which provided a convenient, reliable model for research on different eukaryotic gene regulation sequences.In this study, total RNA was extracted from immunizing silkworm, cDNA was synthesized by RT-PCR reactions and these cDNA was used as templates in PCR amplictions. The PCR products were 340bp, the same length of the predicted cecropinB gene, and was cloned to pMD18-T to analysis its sequence, and was compared with its gene and amino acid sequence in GeneBank. The result showed that the gene and amino acids of cecropinB have very high homology to 100%. The PCR product was linked with pMD18-T and the recombinant vector was named by pMD18-B. The positive recombinant was selected and digested with two restriction enzymes, the pECFP-C₁ vector which had fluorescence report group was digested with same double restriction enzymes, cecropinB gene which had sticky ends were purified and linked with pECFP-C₁ which also had the corresbonding sticky ends and was purified. The positive recombinant was selected finally. By this means, eukaryotic expression vector of cecropinB was constructed successfully. It was named by pECFP-B. Then, the goat mammary epithelial cells was isolated by Collagenase digestion and cultured. And separation of cultured goat mammary epithelial cells was identified using immunohistochemical staining method to determinate the expression of keratin 18 of mammary epithelial cell. Further cationic liposome vectors was used to transfer the pECFP-B into goat mammary epithelial cells,thus the expression ofβ-galactosidase could be detect by two levels-mRNA and protein .The CecropinB protein was expressed successfully in goat mammary epithelial cells by RT-PCR and agrosr diffusion test . The vector contained pECFP-C₁ report gene ,which expressed fluorescin ,so the green fluorescent light could be seen in fluorescence microscope . Furthermore, the vector contained Neor resistance gene. So the goat mammary epithelial cells which were transfected with pECFP-B were screened by G418 of 800mg/mL about 3 days ,and 200mg/mL over 30 days .Steady expression of cecropinB was confirmed by fluorscent test The expression of cecropinB in the transfected cell culture medium and the broken cells liquid at different stages was detected. According to the testing results, in the cell culture medium the expression of cecroinB protein could not be detected while the cell broken liquid had a gradually upward trend by agrose diffusion test from 3d to 30d .the untransfected cells were used as control. The result shows that the cecropinB gene can be transmited to future generations with the cell division. In the heathy goats and the mastitis goats, the recombined plasmid DNA was injeced into udders, the anti-bacterium activity of milk was detected. The result showed CecropinB was expressed in goat udder successively.