Expression of human insulin gene in baby hamster kidney cells and its effect on decreasing blood glucose

Hua LI; Ping WANG; Weiquan LIU; Jigui WANG

ABSTRACT

BACKGROUND:

With the development and application of molecular biology and molecular genetic techniques,people research the production of human insulin by means of genetic engineering,and consider from the molecular level to reconstruct the cloning cell line which excretes insulin,so as to replace insulin injection and islet transplantation to treat diabetes mellitus.

OBJECTIVE:

To construct and screen human insulin gene eukaryon expression carrier of high performance.

DESIGN:

A randomized control experiment.

SETTING:

Experimental Animal Department of China Medical University.MATERIALS Forty healthy adult Kunming mice of clean degree, weighing 20-30 g, half males and half females, were provided by the experimental animal center of Munitions University of Chinese PLA,and they were free to the access of the artificial granule food and water,and all the mice were lighted for 14 hours every day. Escherichia coli DH5α and BHK cell line were preserved by the experimental animal center.

METHODS:

Recombinant plasmid pEF1α-ImINS was constructed with routine method.The baby hamster kidney (BHK) cells were transfected with recombinant plasmid pEF1α-ImINS and other 3 recombinant plasmids of insulin, then screened with G418, and the positive clone cells were passaged to the 20th generation, the expressions of insulin and/or proinsulin in BHK cells were detected with radioimmunoassay and immunohistochemical technique. The PBS suspension (5×107) and the supernatant (0.5 mL) of the 20th generation BHK positive cells trasnfected with pEF1α-ImINS were injected intraperitoneally to each mice, the blood glucose was detected before and after injection to analyze the biological effect of the transgeneic product in decreasing blood glucose.MAIN OUTCOME

MEASURES:

The integration and expression of insulin gene in BHK cells and the changes of blood glucose before and after injection were mainly observed.

RESULTS:

The highest insulin expression was 7.984 mIU/L,the gray value of insulin expression was 177.50±5.10 in the cytoplasm of BHK cells, and 150.30±1.43 in the nuclear. The blood glucose at 6 hours after injection of the suspension of pEF1α-ImINS transfected BHK clone cell strain was obviously decreased,which was very significantly different from that at 24 hours before injection (P ＜ 0.01).

CONCLUSION:

The recombinant plasmid pEF1α-ImINS is the plasmid with the highest expression of insulin in BHK cells.The pEF1α-ImINS transfected clone cell strain has insulin expressions in the mice, and plays a role in decreasing the blood glucose of normal mice.