Clonging And Expression Of Mini Pig Lactoferrin N-lobe Gene

LF (Lactoferrin) is a kind of inartificial glycoprotein linked Fe. It widely exists in outside secretions such as latex, blood, tears, saliva, sweat, semen, hydramnios, pancreatic juice, bile, small intestine liquid, and it is also in blood plasma and neutrophil. There is the most in the latex, especially in the colostrum. LF is a protein of manifold biological activities as the following: antimicrobial activities, antioxidation activities, anticancer, immunoregulatory and so on. In order to know its biological activities well, find out its applications and establish the foundation for later, our studies are as bellow:Test 1. 5g mammary gland tissue was obtained from 15-day lactating sow by surgical. RNA was extracted by Trizol, the Cdna of Porcine Lactoferrin (PLF) was obtained by RT method and PLF-N gene was cloned by RT-PCR method. This gene contains a 1077bp coding sequence of PLF-N. A codon between 1094-1096bp in PLF gene was replaced with a"TAA"stop codon through primers design and PCR. Therefore protein translated from the new ORF contains 359 amino acid residues. Subsequently, this gene was cloned into pMD19-T-simple vector and sequenced. The sequencing results were aligned with the sequence (Genbank, AY306198, EF012241, L77887, M81327, M92089), indicating that the homology between these two sequences was 99%, indicated that this gene is PLF.Test 2. The PCR products, pMD19TS-PLF-N, contains right DNA segment and eukaryocytic expressing vector pET-30a(+) were digested with EcoRⅤ/EcoRⅠ. And then, the right gene segment with EcoRⅤand EcoRⅠtag end and vector segment were abtained, and these two segments were linked using T4 DNA ligase. The ligased products were transduced into DH5αand gene was judged by ligase digestion. The gene expression vetor was transformed into E.coli BL21; the expression was induced with IPTG. The analysis of results indicated that the PLF-N was characteristicly expressed in E.coli. The optimal method for expression protein is the OD600 of E.coli grow in TB is about 0.5, the concentration of IPTG is 1.0 mM and the inducement time is 3h. Test 3. The expressing vector was grown in TB of 30℃, abducted by 1.0 mM...