Study On Expressing Of Lumbrokinase Gene In Cow Mammary Gland

Lumbrokinase(LK), as a new antithrombotic medicine, has been widely used in prevention and treatment of ischaemic cerebrovascular diseases because of its thrombolytics function. At present, it is usually extracted from the earthworms, and it is difficult and time-consuming to purify.In order to solve these problems, we developed a new method to obtain Lumbrokinase by means of gene-engineering. Lumbrokinase was expressed in the milk of cow mammary glands.The expression cassette, including goat β -casein promoter(BCP), LK cDNA and bovine growth hormone(BGH)polyA, was cloned into pLNCL to construct the recombinant retroviral vector pLN-BCP-LK~R. After injecting the recombinant plasmid into lactating mammary glands of cow, LK was shown to be expressed transiently in the milk.The recombinant retroviral vector was transfected into PA317 cells for package by liposome transfection, after 2-weeks of G418 selection, G418-resistant PA317 colonies were obtained and amplified. The supernatant of cell culture was harvested and was used to infect NIH3T3 cells to measure the viral titer of recombinant retrovirus. Results showed that the highest titer of viral supernatant was 1×10~4CFU/ml. The supernatant of cell culture was injected into the mammary gland of pregnant cow, the result of FAPA showed that LK gene had been expressed continuously in the milk of the post partum cow.The milk containing the expression product was separated on SDS-PAGE. The result indicated that the molecular weight of the protein of interest was 40kDa.