Human Lactoferrin Breast Eukaryotic Expression The Carrier Pbc1-hlf Was Constructed,

We constructed the middle vector pGEMEX-I-hLF to add the Sal I sites on the both terminals of the hLF cDNA, then digest the pGEMEX-I-hLF with restriction endonuclease Sal I to obtain the hLF cDNA and digest the vector pBCl with restriction endonuclease Xho I to obtain the liner vector. The expression vector could be constructed by ligation. Then it is used in the following experiments.The middle vector is easer to construct because of its size and directional cloning. But there are some problems in constructing the expression vector: (1) The transfection effect would be lower because the expression vector pBCl-hLF is about 21kb; (2) The target fragment hLF cDNA is much smaller than pBCl, there would be some difficults in ligation; (3) Directional cloning can't be used because there is only one cloning site on pBCl.We tested different conditions to solve those problems: (1) Optimized the preparation of the component bacteria; (2) Optimized recovering the fragments of DNA from agarose gels; (3) Optimized the ligation system; (4) Optimized the dephosphorylation of plasmid DNA; (5) Tested the temperature of ligation.The results show that: (1) The concentration of the nucleic acid is the most important factor. The better result could be obtained when the concentration of the nucleic acid is higher than 100ng/μl and the molar ratio of plasmid vector to insert DNA fragment was approx. 1:3 in the ligation. (2) The purity of the nucleic acid would affect the effect of the transformation. (3) PEG8000 may be harmful to the component bacteria; it would be discarded from the nucleic acid before transforming. The target clone was obtained when the concentration and the purity of the nucleic acid were adjusted and the PEG8000 was discarded.The experiment explored the conditions of constructing the mammary gland expression vectors using pBCl.