猪乳铁蛋白基因的克隆及其重组乳酸菌表达系统构建

Cloning of porcine lactoferrin and construction of expression system in recombinant Lactobacillus

根据猪乳铁蛋白N端基因序列及表达载体质粒的基因融合特点,设计一对引物,以泌乳3日龄母猪乳腺组织RNA为模板,进行RT-PCR,获得含有猪乳铁蛋白基因N端1 077 bp目的片段,将其与表达载体质粒pPG612.1进行连接,通过转化进入宿主菌JM109细胞内,通过质粒提取、PCR鉴定、酶切鉴定和序列测定分析,表明猪乳铁蛋白N端PLFN基因已成功插入到表达载体质粒中,获得了猪乳铁蛋白干酪乳杆菌表达载体质粒pPG612-PLFN。将获得的重组质粒再分别电转化入干酪乳杆菌ATCC393、植物乳杆菌KLDS1.0344、副干酪乳杆菌KLDS1.0652及戊糖乳杆菌KLDS1.0413中,获得表达猪乳铁蛋白基因的多种重组乳酸菌分别为pPG612-PLFN/L.casei,pPG612-PLFN/L.plantarum,pPG612-PLFN/L.paracasei和pPG612-PLFN/L.pentosus.,为猪乳铁蛋白的乳酸菌表达及重组乳酸菌抗菌制剂的制备奠定了基础。

Lactobacillus casei was selected as a bacterial carrier for the expression of the N-lobe of porcine lactoferrin （PLFN）. A 1 077 bp fragement of the gene from PLF was cloned from mammary gland tissue of the lactating sow at the third day by RT-PCR method. It was in accordance with the characters of gene fusion and translational fusions of expression vector plasmids and designed a pair of primers with Oligo6.0 and used the primers to amplify the PLFN gene, the gene was ligated with the vector plasmids pPG612.1 and was transformed into the host strain JM109, and then the gene of PLFN was cloned into the Lactobacillus vectors pPG612.1, resulting in the recombinant expression vector plasmid pPG612-PLFN by plasmids extraction, PCR restriction enzymes digest and sequences analysis. The recombinant plasmid was respectively transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS1.0413 by electroporation, and produced the recombinant strains of pPG612-PLF/L.casei, pPG612-PLF/L.p/antarum, pPG612- PLF/L. paracasei and pPG612-PLFN/L.pentosus, respectively. The result showed that PLFN gene had inserted into the expression vectors and achieved multiple Lactobacillus expression systems. It has elected the base for the expression and the production of biologically active porcine lactoferrin in Lactobacillus.