Cloning And Expression Of Growth Hormone (GH) Gene Of Silver Fox

Growth hormone (GH or somatotropin, ST) is one of the most important hormones which stimulates animal to grow and excrete from pituitary. With the improvement of pituitary GH purification technology and the application of gene recombination, the bovine, pig and chicken GH which have been injected into milch cow, pig, chicken and sheep to develop their productive efficiency have been highly expressed through E . coil system and have informed obvious growth function. The development and born of gene project technology provide new method for mass production of GH. It has played a important role in improving animal growth rate, the using ratio of feed and production return, also in the aspect of economic animal breeding and stock raising. In a view, GH makes a promising application in the aspect of production and practice. According to this, various types of animal GH have been cloned and expressed out of body and researchers imagine farther investigating their biological functions and related use. At present, there is no report about sliver fox growth hormone (fGH) seen in our country. This experiment is to separate fGH gene from sliver fox pituitary issue and clone, express out of body. It will provide the bases on materials and theory for deeply invest on actual application of GH.Our objective is to gain fox growth hormone gene by cloning and expressing and finally to improve progenitive productivity, making more GH excrete from fox and increase its coat in size by injecting or feeding GH. The traditional gene project method was used and successfully constructed eukaryotic expression plasmid pcDNA3.1-fGH and prokaryotic expression plasmids pPROEXTMHTa-fGH. The result showed that both of them had expressed in the cell. It is significant in empoldering growth medicine and father study on possibility of fox GH gene therapy. At present, there is no report about sliver fox growth hormone (fGH) seen in our country. We separated fGH gene from sliver fox pituitary issue and clone, express out of body. It will provide the bases on materials and theory for deeply invest on actual application of GH.In order to gain biological functional fox growth hormone and deeply study its function and application, we constructed prokaryotic expressed plasmid of pPROEX ' HTa-fGH transferred into E.coil and eukaryotic expressed plasmid of pcDNA3.1-fGH transfected into male rat. Then the mature protein was got successfully expressed. The method is that total RNA from fox pituitary issue is picked up with Trizol Kit. The specific primers of fGH gene which based on the sequence of the other canine strains was designed and synthesized. Specific fGH cDNA. 700bp, was amplified by RT-PCR with genomic RNA of fGH strain as the template for cDNA synthesis. PCR products were cloned into pMD18-T vector and named as pMD18-T-fGH. In order to express fGH gene in eukaryotic cell, according to the gene sequence of canine GH in Gene Bank, we designed a pair of primers c1, C2. The up primer c1 includes the 5' region homologous sequence and the point of HindIII restriction enzyme; the down primer includes the 3' region homologous sequence and the point of EcoRI restrictionenzyme. A 700bp-band was got by PCR with template of pMD18-T-fGH, after digested by restrict enzyme Hindlll and EcoRI. Then we ligated it into eukaryotic expressed vector pcDNA3.1 which digested by same enzyme. After identified by enzyme digestion and PCR, we transfected the positive recombinant eukaryotic expressed plasmid into calf muscles of male rats with following concentrations: 10u.g/100ul, lOOug/lOOul and 250ug/100ul Comparing with the rats which transfected pcDNA3.1 vector with nothing, we found the recombinant plasmid expressed through took the muscle issues of rats and picked up the total RNA then PCR every six days. Contemporary, we sub-cloned fGH gene cDNA segment with another special primer including the point of BamHI restriction enzyme by PCR and recombined it into prokaryotic vector pPROEX?HTa which was digested by restrict enzyme EcoRI and BamHI. After ligated, the prokaryotic expressed plasmid pPROEX?HTa-fGH was identified by enzymes digestion and PCR. Then the positive recombinant prokaryotic expressed plasmid was transferred into E.coil DH5 a . Under the inducement of final concentration lmmol/L IPTG, the high expressed amount of GH protein was detected by SDS-PAGE electrophoresis and it occupied total E.coil protein 35%.The recombinant protein in Ecoil DH5 a expressed in the form of excuse body. Cloning of fox GH gene and the expression whether in prokaryotic or eukaryotic system lay a foundation of farther study on the structure, biological function even operated machismo of fGH gene on the level of cell and molecule. In this way, we can make a deep research on the practice aspect of promoting growth in mammiferous GH gene.