Construction And Cell Expression Eukaryotic Expressional Vector Of Human LL-37 Gene

Mammary gland bioreactor is a technology of product transgenic cows which inserted exogenous gene into the cow mammary gland of organ-specific expression. It has a wide range of potential value of specifictiy, stability, high efficiency in food and drugs, medical, industrial production and livestock breeding industries. Firstly, cows’ breasts are an organ of natural and efficient protein synthesis, which can reduce the damage of transgenic cows with excellent permeability barrier function limits the foreign gene into the systemic circulation. Secondly, the high milk yield of cows can continually produce human therapeutic proteins who needed. In addition, cows mammary gland bioreactor can also change the composition of the milk, making it closer to human milk in a higher value field.Antimicrobial peptide is a small molecule peptide which widely present in the human body, animals, plants, insects. Which is one of an important component of the body’s natural immunity. Antimicrobial peptide can easy to produce drug resistance with broad-spectrum antimicrobial activity against bacteria, fungi, viruses and tumor cells were antagonistic effects. Many bacteria have a significant resistance with long-term effects of penicillin, streptomycin and other traditional antibiotics. The study found that endogenous antimicrobial peptide attracted the attention of many scholars because of its safe and effective features, become a new weapon against pathogenic microorganisms to replace traditional antibiotics.hCAP-18(human Cathelicidin Antimicrobial Peptide 18KDa) is the only member of the tree in the human body’s antimicrobial Peptides Cathelicidin, released by the enzyme interpretation C-terminal fregment is called LL-37. Also known as LL-37/ hCAP-18, Which named before its N-terminal two amino acid residues leucine(L) and the total number of amino acid residues. LL-37, except with anti-microbial function, but also has a neutralizing endotoxin and immune regulation and role widely distributed in the body, researching open-minded such as infectious diseases, autoimmune diseaese, new drug development, clinical and so on in the future.Therefore, this study researched for the target to carry out the development of new therapeutic proteins based on molecular biology. Construction of pc DNA3.1-LL-37 eukaryotic expression vector and text its expression in bovine mammary epithelial cells and fetal fibroblasts. In this study, amplified gene fragment of LL-37 from human blood samples by RT-PCR, which connected to the pc DNA3.1-His-V5(+) vector, The recombinant plasmid defined as pc DNA3.1-LL-37 was testified by single Xho I restriction enzyme digestion. Then, the recombinant plasmid was transferred into the bovine mammary epithelial cells and fetal fibroblasts with liposome and was screened by G418. RT-PCR and Western Blot were applied to check the expressions of the plasmid at transcription and translation levels, respectively. The 523 bp of LL-37 gene was obtained from the post-transfection cells by RT-PCR. The m RNA expression of LL-37 gene was found in different period by q RT-PCR of two cells, the highest expression level was found at stability transfection while 17 k Da specific band was obtained by Western Blot. Results showed that the LL-37 gene was successfully inserted into the chromosome of bovine mammary epithelial cells and fetal fibroblasts, and fetal fibroblasts high expression of protein than cow mammary epithelial cells which provided the foundations for the construction of LL-37 protein secretion to cow mammary gland bioreactor.