Xuhuai Goat H-FABP Gene Cloning And Preparation Of Transgenic Mice And Transgenic Goat

The Fatty acid binding protein family (FABPs)which belong to the apolipoprotein superfamily is one kind of small molecular weight proteins, the weight of protein is about12-16kDa.The protein is also one kind of fatty acid transport protein. This protein family were earliest found by Ocker worked in University of California in1971. He found the protein family when he reserched the regulation of fatty acid absorption in the rat small intestine mucosa. So far,11subtype have been found and named according to the main distribution organs or tissues.They are intestinal type (Intestinal, the I-FABP), heart-type (H-FABP), liver type (L-FABP), adipocyte (A-FABP), brain type (brain FABP), epidermal type (epidermal FABP), ileal-type (I leal FABP), medullary phospholipid type (myelin FABP), kidney-type (renal FABP) the testicles type (T-FABP), fatty acid binding protein and cellular retinol binding protein (CRBP) and cellular retinoic alkyd binding protein (CRABP). There are strongly homology between the different subtypes. The protein family account for3%-8%of the total soluble protein in the cells. The main function is taking part in the intracellular transportation of fatty acids. The protein family regulate fatty acid metabolism by transporting fatty acids from the cell membrane to the synthesis of triglycerides and phospholipids.Heart-type fatty acid binding protein is the most widely distributed and the most important subtype among the total protein family. It exists primarily in three kinds of cells:high βoxidation rate cells (such as cardiac, skeletal muscle cells);(2) high strength fat metabolism, especially steroidogenic strongly cells (such as the endocrine system of the adrenal cortex cells, lactating mammary gland);(3) mesodermal origincells. The protein most abundant in the myocardium, accounting for4%-8%of all soluble protein in the heart. Its main function is to prevent the intracellular accumulation of free fatty acids. The protein is one kind of soluble cytoplasmic protein with a molecular weight of14-15KDa.H-FABP is an acidic protein which has a strong conservative among different species. The structure of human H-FABPcomposed of132amino acid residues while cattle, goat, wild boar, small house mice consist of133amino acid residues. In this study, We cloned Xuhuai goat Ⅱ-FABF gene CDS and anaiysised its sequences information. The results showed that:Xuhuai goat H-FABP gene CDS contained402bp, encoding133amino acids. The protein molecular size was14.76kDa and isoelectric point was6.369. It was also an acidic protein. There were high similarity of H-FABP gene sequence and amino acid sequence between Xuhuai goat and chicken, Rattus norvegicus, cows, wild pigs, donkeys and zebra fish which reported on the GenBank. The protein there is not the signal peptide and sugar-free glycosylation sites,but it contained eight phosphorylation sites. According to the two kinds detection method of comprehensive protein(GFP) and indirect immunofluorescence, we confirmed that Xuhuai goat H-FABP protein expressed in the cytoplasm. We transfered recombinant vector carried the H-FABP of Xuhuai goat into male mice testicle by testicular injection,and then put male and female mice into one cage.After GO genetion gave birth, we detected the target gene on DNA and protein levels. At last,we got4%(7/175) positive individuals. In the same way,we detected the G1generation gave birth by GO Positive generation,and got30.23%(13/43) positive individuals. Myocardium, leg muscle and longissimus muscle of GO and G1positive individuals were collected and determined the intramuscular fat content. The results showed the intramuscular fat content of positive individuals was significantly higher than that of negative individuals. The biological functions of the exogenous gene expressed fully. We prepared transgenic goat used the methord of microinjection. We transfered Xuhuai goat H-FABP gene into Ru Boer goat fibroblast cells and got positive cell lines by G418screening. As nuclear donor, positive cells were injected into mature nuclear oocyte. We transfered recombinant embryo into receptive ewe uterus, and four case of positive aborted fetuses and one positive individual survival obtained, positive ratio were83.33%.In line with the basic methord of praparation of transgenic mice, we produced transgenic goat by testicular injection, and got17F1individuals in which there were3positive individuals,positive ratio were17.65%.