Construction And Primary Expression Of Human Osteoprotegerin In Mammalian Vectors

Osteoprotegerin(OPG) is the factor that has important negative effect on the differenciation and development of osteoclast, and play the important role in the pathogenic mechanism of many deseases associated with bone metoblism. But the much more mechanism of its effect during the couse of physiology and pathology should be further research. For this reason,large amount of OPG produced by engineering can meet the demond of basis and clinical application research.In this research, after the complete coded gene of the human OPG cDNA was amplified and cloned into the mammalia vector pcDNA3.1/CT-GFP, the recombination OPG expressing vector pcDNA3.1/OPG-GFP" has been constructed. The recombinative vector was transfected in COS₇ cell and performed transient expression. The function of the vector would be verified by ELISA and Western blot. Furthermore, the vector pcDNA3.1/CT-GFP has been modified by means of deleting the enhancer sequence of the CMV early promoter that drive the exogenetic gene expressing. The opg cDNA was cloned into the new vector, and the new OPG expressing vector pcNDA-CMV^T/OPG-GFP^- has been constructed. The amount of opg expression would be compared between the two OPG recombinative vector.In order to express OPG by high efficiency and stabilization, and quickly screenthe high expression cell strain from the CHO cell that has the character of dihydrofolate reductase deficiency (CHO-dhfr"), the DHFR expression unit carrying weak promoter would be inserted in the vector pcDNA3.1/OPG-GFP". The DHFR expression unit was constructed by means of PCR mutant tecnique. After amplified respectively by primers added complementary sequence at the 5' terminal, the three fragments including the weakened SV40 promoter , dhfr cDNA coded gene sequency and SV40 polyadenylation sequence were overlapped and extended to DHFR expression uint. While the unit was cloned into the vector pcDNA3.1/OPG-GFP", the vector would acquire a new choice and marking gene(dhfr). The new vector was linearized and transfected CHO-dhfr" cell. At first, the cells were cultured in methotrexate (MTX) -free medium, in order to screen out the cell strain of expressing purpose gene. Then the positive cell strain would be cultured in gradient concentration MTX to screen the cell stain that can efficiently and stablily expressing OPG .The vector pcDNA3.1/OPG-GFP" has the function of expressing opg, which was virified by ELISA and Wstern blot. And in the amount of expressing OPG there is no difference between the vector pcDNA3.1/CT-GFP and the vector deleted the enhancer sequence of the CMV early promoter.The concentration was up to 200⁴00ng/ml. After the DHFR expressing unit was cloned into vector pcDNA3.1/OPG-GFP'and the new vector was linearized and transfected the CHO-dhfr- cell, the cell was verified that DHFR and OPG cloned gene has been integrated into genome DNA by respectively extracting genome DNA and total RNA to PCR and RT-PCR, Furthermore, under the effect of gradient MTX, we can see that with the concentration of MTX increasing , the amount of OPG expression constantly ascending from the basic expression 50¹00ng/ml up to 900 ng/ml.