Expression Of Human Placental Alkaline Phosphatase In Hen's Oviduct And Other Cell Lines

A large and rapidly increasing number of proteins are required in medicine and industry in kilogram or metric ton amounts per year. Many of these proteins assume a native conformation only in the correct cellular environment and require specific post-translational modification. Bacteria, yeast, and insect protein express systems couldn't meet these requires. A number of protein, including biopharmacerticals, have been produced in the milk of transgenic mice, rabbits, pigs, sheep, goats, and dairy cattle at concentrations of up to ten grams per liter. Although promising, mammary gland bioreactors have several drawbacks, including long generation times. The biochemical complexity of milk complicates the purification of recombinant proteins. So many investigators engaged in the studies of chicken egg bioreactor.In this studies, human placental alkaline phosphatase, a kind of glycoprotein, expressed in transgenic chicken oviduct, primary cultured chicken embryo cells, human breast cancer cells, and human colon cancer cells. We investigate the expression of human glycoprotein and promoter activities in transgenic oviduct, chick embryo cells, human colon cancer cells and human breast cancer cells.Two kinds of expression plasmids were used in this study. One is chick oviduct tissue-specific expression plasmid pAB-Sig-SV40. The chimeric plasmid pAB-Sig-SV40 is consist of ovalbumin gene promoter, the first intron of the ovalbumin gene , and SV40 T antigen transcription terminator. The other is pSEAP2-control plasmid expressed under the regulation of SV40 early region promoter and SV40 enhancer. pSEAP2-control was used as a control plasmid in our experiment.Human cancer cell MCF-7 was derived from a pleural effusion and was characterized to possess estrogen receptors. Human colon cancer cells don't has estrogen receptors. Growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10-8M estrodiol, penicillin at 100 units/ml, and streptomycin at 100 ug/ml, cells were grown at 37 癈 in a humidified 5% CO2-95% air incubator.1 The amplification plap gene and expression in Escherichia coli cellsThe coding sequence of the gene of human placental alkaline phosphatase(plap) was amplified from the pSEAP2-control plasmid by polymerase chain reaction(PCR) method,This plap gene was inserted in-frame in the BamHI and XhoI site of pSK plasmid and was sent to be sequenced. the correct sequence of plap gene was cloned into pET-32a plasmid to express in Escherichia coli cells.2 The production of multiclone antibody in mouseRecombinant PLAP protein expressed in Escherichia coli cells, and was judged to be what we wanted, the recombinant protein was injected into mouse's abdomen, after 3 times injection of the proteim, antibody was produced in serum. Western blotting method was used to detect the production of antibody in serum.3 The construction of pAB-Sig-p/ap-SV40 plasmidPlap gene in pET-32a-plap which express recombinant proteins was digested by restriction endonuclease and was inserted in the BamHI and XhoI site of chick oviduct tissue-specific expression plasmid pAB-Sig-SV40 and oriented such that the 3'terminus of the gene was nearest to the unique Xhol site in the plasmid and the expression of this human gene would be promoted and regulated by the chicken regulated sequences. This resultant plasmid has been designated as pAB-Sig-p/ap-SV40.4 The production of transgenic chickenIn order to study the expression of the PLAP in laying hens oviduct, the recombinant plasmid pAB-Sig-p/ap-SV40 were linearized by restriction endonuclease Kpnl before being used for transformation experiments, Other Kpnl sites in pAB-Sig-SV40 and plap gene are not shown. The linearizied plasmid was purified after digestion, and its concentration was measured.Sperm-mediated gene transfer technique was used in this study. The linearized DNA was embeded with liposome as a ratio of 100ug DNA per milliliter liposome. The embeded DNA was mixed gently with fresh sperms which was rinsed and su...