Expression Of Lumbrokinase Gene In Cow Mammary Gland And Construction Of Transgenic Rabbit

Cerebrovascular disease is one of the principal diseases harmful to human lifeand health which shows driving rising tendency. It is very important and significant tofind new medicine to prevent cerebrovascular diseases. Lumbrokinase(LK), as a newantithrombotic medicine, has been widely used in prevention and treatment ofischaemic cerebrovascular diseases because of its thrombolytics function. At present, itis usually extracted from the earthworms and is difficult and time-consuming to bepurifed. Animal mammary gland biology reactor belongs to transgenic animal, and itscentral content is highly expression of exogenous gene driven by specific promoter inanimal mammary gland, and to produce target protein with high added value in milk.Retrovirus-mediated gene transfer technique is one of the highly efficient genetransfer methods established in 1980. By recombination with exogenous gene andretrovirus vector and package into cell line, a defected recombinant retrovirus wasproduced and target cell could be transfected successfully, exogenous gene could beintegrated into the genome of target cell and expressed in it. In order to solve theseproblems, we developed a new method to obtain Lumbrokinase by LK transgenicrabbit production with retrovirus-stem spermatogonium transfection system. It willprovide an important evidence for mammary gland reactor production.The mammary specific expression vector pBLK and recombinant retroviralvector pLN-BCP-LKR with expression cassette, goat β-casein promoter (BCP) and LKcDNA, were constructed. The recombinant plasmids were dissolved into 20mL sterilephysiological saline on dose of 200ug, 500ug, 1000ug and 2000ug, respectively. Thesolutions above were injected into mammilla duct of milked cows with identicallactation in stable lactation period. The mammary gland tissue was infected and milksamples in different stages were collected. The fibrinolysis activity was detected bythe diameter of fibrin flat plate agarose with fibrin agarose plate assay. The resultsshowed that BCP was expressed transiently in the milk and fibrinolysis activity ofmilk samples appeared after 3hs of infection, peaked at 6-16hs, and lasted until40-60hs. There are significant differences between 500ug and 200ug, 1000ug and2000ug(P<0.05) and no significant differences among 200ug, 1000ug, 2000ug (p>0.05) in pBLK plasmid injection groups. There are significant differences between500ug and 1000ug and no differences among other dose in pLNBCPLK injectitongroups. Between two plasmid treatment groups, there is significant difference between500ug and 1000ug, no significant difference between 200ug and 2000ug. The resultsshowed that the best treatment dosage is 500ug plasmid.The recombinant retroviral vector was transfected into PA317 cells for packageby liposome transfection, after 2-weeks of G418 selection, 5 strains of G418-resistantPA317 colonies (P1, P2, P3, P4, P5) were obtained and amplified. The supernatant ofcell culture was harvested and was used to infect NIH3T3 cells to measure the viraltiter of recombinant retrovirus. Results showed that the highest titer of viralsupernatant was 1×104CFU/ml. The supernatant of cell culture was collected andstored at -80℃. 80ml virus containing solution was unfreezed and then injected intothe mammary gland of pregnant cows, and target protein expression was detectedweekly. The result of FAPA showed that LK gene had been expressed continuously inthe milk of postpartum cows and lasted about 112ds.The milk containing expression products was separated on SDS-PAGE. Theresult indicated that the molecular weight of the target protein was 43kDa. The resultsof lamellar scaning analysis showed that the transient expression level of thelumbrokinase was 350mg/L in pBLK treatment groups and 320mg/L in pLNBCPLKtreatment groups, the LK stable expression level was 180mg/L.In order to study the gene transfering result mediated by recombinant retrovirus-spermatogonial stem cells, recombinant retroviruses, together with typan blue andPolybrene, were co-injected into convoluted seminiferous tubule of 7 male rabbit testis(3-months) to infect spermatogonial stem cells in vivo. When sexual maturity of the 7rabbits, histological section of 2 rabbit testis was observed and other 5 rabbits werepropagated. Results indicated that there are no pathological changes in testis, liver,spleen, lung and kidney in two rabbits.The two individuals in F0 generation from otherfive rabbit showed LK gene positive by PCR amplification. Among the 13 individualsof F1 generation, 2 of 8 in one family were positive and all in the other family werenegative by PCR and southern blotting detection. Results indicated the positive rate ofcollected transgenic rabbits is 6.25%, and recombinant LK retrovirus was no harmfulto main organs of rabbit, which could grow, develop and breed normally.All the results above indicated LK gene could be transient and lasting expressionin cow mammary gland, and transgenic rabbits had been obtained. All these resultsprovided evidences theoretically and practicallly for transgenic cow mammary glandreactor production.