Construction And Expression Of Rat, Mouse And Pig DHRS4 Prokaryotic Expression Vectors And Preparation Of Polyclonal Antibody Against Mouse DHRS4

Objective:NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) is an important retinol dehydrogenase/reductase enzyme in the atRA synthesis.in vivo ,atRA is derived from vitamin A or retinol through a two-step oxidative metabolism reaction and retinal is the intermediate product.NADP(H)-dependent retinol dehydrogenase/reductase plays an important role in the reaction of Retinol changing into Retinal.NRDR gene is localized on 14q11.2, consisting of three copies, namely DHRS4, DHRS4L2 and DHRS4L1. However, in most other mammals there is only a single copy of DHRS4.For example, rats, mice, pigs, cattle, horses, and dogs. in a few of other mammals there are two copys .They are DHRS4 and DHRS4L1. For example, chimpanzees, apes .Because NRDR is a complex regulation of expression in humans,so the single copy in mice as a model research can help us to further explore the regulation of expression of the human body NRDR.Therefore, DHRS4 prokaryotic expression vector and the preparation of mouse polyclonal antibody-based research can help to study mouse NRDR.For the function and activity of NADP(H)-Dependent Retinol Dehydrogenase/Reductase (NRDR),our group has studied the role of NRDR in rabbits and found that NRDR was involved in oxidoreduction between retinol and retinal with high activity. NRDR can not only reduce retinal to retinol but also show relatively broad reductive activities to some other materials, which is similar to retinol and retinal.In recent years, it was reported that activity of the enzyme was different in the different animals, but the role of NRDR in the human body has not been reported。Therefore, it is necessary to in-depth study their physiological functions and their differences. However,Low content of the enzyme in the body, so separation and purification is difficult. In vitro expression of enzyme we can get a large number of recombinant enzyme ,which provides experimental materials for regulation and expression of DHRS4 gene and function study.Methods:the open reading frame (ORF) of DHRS4 gene was amplified from liver and testis of rat, mouse and pig by the RT-PCR. After sequencing, the products were cloned into pDONR^TM201 vector.Then the products were cloned into pDEST^TM 17 vector by LR recombination reaction .After identification by DNA sequence analysis , the recombinant piasmids were transformed into component E.coli BL21-AI cells and the pDEST^TM 17 /DHRS4 fusion protein was expressed with the L-Arabinose induction .The anti-DHRS4 polyclonal antibody was produced by immunizating the rabbit with the mouse fusion protein. The resultant antibody was purified by AKTA primer and to detect DHRS4 expresstion of mouse by western blottingResults:The recombinant vectors pDEST^TM 17 /DHRS4 of the rat,mouse,pig were successfully constructed .pDEST^TM 17 /DHRS4 fusion protein was expressed abundantly at 4 h after L-Arabinose induction and anti- DHRS4 polyclonal antibodies of mouse were obtained . Western blot analysis demonstrated that the antibodies specifically detected DHRS4 expresstion in mouse liver and testis .Conclusions:We have succesfully constructed pDEST^TM 17 /DHRS4 recombinant vectors of rat,mouse and pig and obtainted anti- DHRS4 polyclonal antibody of mouse.