Cloning Of Human Lactoferrin Gene And Constructing Of The Expression Vectors

Human lactoferrin, also named lactotransferrin, is a bioactive, versatile protein, and has large potential in nutritional and therapeutic applications. Theoretically speaking the galactophore tissue of animals is the best apparatus to express hLTF. However, one major drawback in the research of animal mammary gland bioreactors is the low level of foreign gene expression in the milk of transgenic animals. Therefore, to obtain high and stable expression of foreign gene, the various promoters have been used with constructing expression vector in recent researches.First, the 2.3kb cDNA of human lactoferrin sequence was obtained by high fidelity polymerase chain reaction (PCR) and inserted into pMD19-T vector subsequently. Then it was identified by enzyme digesting, PCR and sequencing. The sequencing indicated that the homology of human lactoferritin cDNA sequence was 99.5% comparing with those of GenBank, but no amino acid replacement. The human lactoferritin cDNA was subcloned into the reconstructed pcDNA3.0(pA), the correct vector was identified by enzyme digesting, PCR and sequencing. We successfully cloned hLTF and constructed the recombinant eukaryon expression vector pA-hLTF. Then the rabbits were immunized by muscule injection of the pA-hLTF. After immunized fourth, test the titer of the antibody in the serum by indirect ELISA. The result showed that the titer of the antibody reach 1:1600.Then the 3.5kb and 4.6kb regulatory region of the WAP5'and WAP3'were obtained by high fidelity PCR, applied to mouse genome, and inserted into pMD19-T vector subsequently. After verifying correctly they were excised and inserted into pcDNA3.0 with hLTF in vitro to form pW2-hLTF. ThepW2-hLTF was testified by enzyme digesting, PCR and agarose electrophoresis. The results showed that the mammary gland-specific expression vector pW2-hLTF was successfully established.