Construction Of Expression Vector For Human Thrombopoietin In Mammary Gland

Construction of mammary gland bioreactor is the key to choose suitable promoter and foreign gene, which is the milk protein gene, the foreign gene can be high-effective, specific expression in breast cells. Casein is the main protein in the milk of mammals, widely existed in ruminant and rodents animals. Theβ-casein is one of high-effective expression and contention in the milk protein. In the milk protein of mice and cow respectively contented about 20%, 27%,and 20～30% in other mammals. In the stimulation of galactin, theβ-casein has strongly express-activeness, which regulation element can guide the target gene high-efficiency expression in breast tissue.In order to construction of mammary gland expression-vector, we respectively obtained the bovineβ-casein (bβ-casein)of Holstein cow , mouseβ-casein (mβ-casein)of C57/B6 mice as the promoter, human Thrombopoietin(hTPO) of Chinese as foreign gene . We linked the bβ-casein, mβ-casein and hTPO gene with T- vector (pMD-19 vector), respectively .Then we used theαcomplementary action blue - white spot filtration method filtrated positive cloning.The result of the positive bacterium liquid sequencing showed that the identity of genome sequencing was over 94% which contrasted with the published in NCBI, it can be carry out the next experiment.We cut the bβ-casein, mβ-casein and hTPO gene from the T- vector, which used the restriction endonucl- ease digestion, then we reclaimed and sublimated it using the Agarose Gel Purification Kit. pcDNA3.1 (+) vector had the same restriction endonuclease site to bβ-casein, mβ-casein and hTPO gene ,and then linked it with them using T4 DNA ligase and we identificated it using PCR and restricttion endonuclease digestion, finally we will be link promoter (bβ-casein, mβ-casein) , the foreign gene(hTPO) with the pcDNA3.1 (+)vector, constructed the high-effective, specific expression-vector to provide condition for the expression of the target gene . We got the results as follow:1. As promoter we cloned about 7.8Kb bβ-casein and about 4.8 Kb mβ-casein gene, respectively. As foreign gene we cloned about 6.1Kb hTPO.2. The result of sequencing showed that the identity of genome sequencing was over 94 % which contrasted with the published in NCBI, it can be carry out the next experiment.3. We cut target gene from the T-vector using restriction endonuclease digestion, and then linkes the target gene with the pcDNA3.1 (+) vector using T4 DNA ligase to construction of specific expression-vector.