| Oxytocin(OT) , a neurohypophyseal nonpeptide ,has both peripheral and central action .Peripherally, as a hormone , OT plays an important role involved in parturition .lactation and regulation of water or sodium . In the brain , OT has relationship with mating and maternal behaviors , study and memory , and relieving pain .Some study found that the concentration of OT in the hippocampus of elderly dementia patients increased. Furthermore , recent study demonstrated OT may inhibit the tolerance and dependence of morphine drugs. All these show that OT plays an important role both in physiological and pathophysiological function in the brain.In peripheral tissue ,OT binds to OTR and then activates multiple signal pathways to exerts its functions .For example ,in uterine smooth muscle cell ,OTR may increases the Ca2+ concentration inside the cell via activation Gq-PLC pathway, then uterine smooth muscle contracts. But the signal transduction mechanism and function of OTR in the brain is unknown .It is very necessary to study the structure of OTR and its signal pathway in the brain .All these study may be helpful to explore the function of OTR in the brain ,also helpful to elucidate the pathogenesis of some disease concerning with OTR.In 1992 , Kimura first cloned the OTR cDNA from human myometrium cDNA library by expression cloning .The full length of OTR cDNA was4.1kb,and the encoding region of the cDNA was 1167 bp. So the cDNA encoded 388-amino-acid protein. Subsequently, the OTR cDNA of other mammalian animals was also cloned .All these clone were from peripheral tissue and cell lines , and demonstrated these clone encoded one type OTR .Some studies show that there is another type OTRin CNS, and OT may exerts different functions like peripheral tissue. The cloning of full length OTR cDNA in the brain was not reported. In 1995, Adan R.A.H. cloned the 290bp encoded the fifth transmembrane domain between sixth transmembrane domain from rat brain by RT-PCR. The DNA sequence of this segment was the same to the uterine .But the gene of this domain is ahead of the third intron in the OTR gene ,so may exist the OTR subtype produced by splicing the third intron. Therefore ,it is necessary to clone the full length of the brain OTR cDNA.We cloned the full length OTR cDNA. both of the brain and uterus by RT-PCR, then we compare their DNA sequence to find any difference .The OTR cDNA is very important to study OTR at molecular level. So the OTR cDNA we have cloned is the base to study the structure , function and its clinical significance.There is no report of OTR gene expression in E.coli system yet. Because we want to get a lot of OTR protein by genetic engineering .So we attempted to express OTR in E.coli BL21 after cloning OTR cDNA. In this study we constructed these prokaryotic expression vectors: pET-28- a -OTR to express the fusion protein of full length OTR and His tag ,pGEX-4T-3-OTRN140,pGEX-4T-3-OTRN40 and pGEX-4T-3-OTRC55 to express the fusion protein of different OTR domain and GST. Then we expressed these cloning plasmid in E.coli BL21 .CHO cell is widely used in the study of protein expression in eurcaryotic cell and signal transduction .So, another aim is to establish a cell line expressing OTR by cell culture and transfection technique .In this study ,we established CHO-OTR cell which expression OTR. Furthermore , we demonstrated OTR expression by western blot.In addition ,we produced OTR antiserum by routine immunization and DNA vaccine technique . In this study ,we immunized BALB/c mouse by pcDNA3.1-OTR and fusion protein GST-OTRN40 . The specificity of immunoreactivity is verified by the western blot analysis . The main results of our research are as follows:1. The rat OTR cDNA full length was successfully cloned by RT-PCR from rat brain and uterus. The full encoding sequence of OTR cDNA both from the brain and from the uterus is identical. Their size is 1167bp.2. We utilized pET-28-a vector to carry the full length of encodin... |
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