Study Of H-FABP Gene And PERV Gene Of Tibet Mini-pigs

Mini-pig,extremely similar to mankind in such aspects as anatomy,physiologic characteristic,nutrition and metabolism,has been already used as common experimental animal to study human diseases.As experimental animal,the mini-pig has already been applied to a lot of biomedical research such as tumour, cardiovascular disease,diabetes,surgery,dentistry,skin burn,blood disease, hereditary disease,nutrition metabolic disease and safety evaluation of new drugs, etc..The strains of mini-pig in China are mainly as follows:Wuzhishan mini-pig, Guizhou mini-pig,Bama mini-pig of Guangxi,Banna mini-pig inbred line,Tibet mini-pig,etc.Because the mini-pig originates in the remote mountain area,they formed mini-pig inbred colony,which is characterized by heredity steady phenotype. Lacking of mini-pig resources,hybridization of a variety of pig breeds generally applied in foreign countries.However this led to mini-pig's big size,variety of coat color,unsteady heredity and phenotype.Tibet mini-pig is one kind of pig breeds that can adapt to the abominable climate of the plateau and adversial environment.Because of natural and historical reasons, the Tibet mini-pigs have kept pure breed resources relatively,possessing the characteristics of appearance and unique biological characteristic different from other pig breeds.The adult Tibet mini-pigs' weight average from 25 to 40 kg.Compared with ordinary pigs,Tibet mini-pigs have such characteristics:small size,the durable compactness of physique,thin skin,tiny muscle fibre,high intramuscular fat,good meat quality.Because of their small size,they are very suitable for studying animal experiment.To use Tibet mini-pig to cultivate standardized experimental animal, laboratory animal center of southern medical university introduced 50 Tibet mini-pigs from Tibet Autonomous Region in 2004,through cultivation of more than four years and scientific management,the Tibet mini-pigs have already adapted to the subtropical climate environment completely,their productive performance and reproductive performance were improved significantly.Their laboratory rearing had been achieved.This article studied the application of Tibet mini-pigs on animal husbandry and biomedical research and the investigation are as follows.First part The H-FABP gene studies of Tibet mini-pigsBackground:The meat quality of domestic pig breed is superior to the introduced pig strain,and there are direct relations in the content of intramuscular fat(IMF).IMF is a major determinant of the quality of pork.The IMF content of domestic pig breed is all above 3%,the high one can be up to 6%-7%,and the IMF content of introduced pig breed is generally lower than 3%.The determination of IMF content in vivo is difficult,even by using muscle samples,we must slaughter pig and take sample after the pig reaches certain weight,and this is waste of time and fund. Moreover,there is blindness to the selection of pig IMF by using traditional method, cycle is relatively long.As a result,looking for the main gene in quantity trait loci(QTL) of influencing IMF content becomes the focus that people pay close attention to extensively.The foreign breeding expert discovers that the heart fatty acid-binding protein(H-FABP) gene is a candidate gene of IMF,so,the studies on genetic variation of H-FABP gene have important actual meanings to the improvement of pork quality.Methods:Collecting 30 Tibet mini-pigs randomly.Under animal's sober state, fixed it,collected 5ml of blood from anterior vena cava,EDTA was added to blood as an anticoagulant.Then blood sample were kept in -70℃low-temperature refrigerator. Blood genomic DNA was extracted using kit(TIANGEN Company).Two pairs of primers was designed to carry out two PCR reactions,named PCR1 and PCR2 respectively.And the position of PCR1 and PCR2 products locate in 5'upstream region and second intron region respectively.The length of PCR1 products is 693bp, the length of PCR2 products is 816bp.5μl of each reaction was then separated by gel electrophoresis in a 2%agarose gel at voltage 80V for 40 minutes.Electrophoresis result was visualized by GelDoc system.Then RFLP analysis was carried on.PCR1 products were digested with HinfⅠ,PCR2 products were digested with HaeⅢ, HinfⅠ* respectively.All restriction endonuclease digestions were performed at 37℃for 6 hours,products were then separated by gel electrophoresis in a 2%agarose gel at voltage 80Ⅴfor 1h.The electrophoretic patterns of the digested material were recorded digitally using a gel documentation system.The H-FABP fragment that PCR1 amplified has four HinfⅠrestriction enzyme cutting site,producing 339,172, 98,59,25bp fragments,1321bp enzyme cutting site is polymorphism site,when it is disappeared because of variation,172 and 59bp fragments amalgamate which produce 231bp,define H allele(339+172+98+59+25bp) and h allele (339+231+98+25bp).The H-FABP fragment that PCR2 amplified has three HaeⅢrestriction enzyme cutting site,producing 405,278,117,16bp fragments,1811bp enzyme cutting site is polymorphism site,when it is disappeared because of variation, 278 and 405bp fragments amalgamate which produce 683bp,define D allele (683+117+16bp) and d allele(405+278+117+16bp).The H-FABP fragment that PCR2 amplified has three HinfⅠ* restriction enzyme cutting site,when three cutting sites all exist,we define it B allele(521+217+47+32bp),producing 339,172,98,59, 25bp fragments,1321bp enzyme cutting site is polymorphism site,when polymorphism site on 1968bp is disappeared we define it b allele(521+264+32bp). Calculate gene frequency and genotype frequency,PIC according to the experiment result,and carry out the significance test of difference of genotype frequency(chi-square goodness of fit test).Calculation of gene frequency, significance test of difference of genotype frequency(chi-square goodness of fit test) was carried out using SAS9.0 software.The procedure code was designed by myself. Polymorphism information content(PIC) calculation was carried out using PIC_CALC software(version 0.6,download from www.bbioo.com)Results:(1)In HinfⅠ-RFLP site of 5'upstream region,the Tibet mini-pigs have polymorphism,the frequency of allele h is 0.80;(2)In HaeⅢ-RFLP site of second intron region,all Tibet mini-pigs genotype are shown as DD genotype;(3)In HinfⅠ*-RFLP site of second intron region,except that one pig is shown as bb genotype,other pigs are all shown as BB genotype,the frequency of allele B is 0.97; (4)Except HaeⅢ-RFLP site,all Tibet mini-pigs are in Hardy-Weinberg disequilibrium state in other sites;(5)The polymorphism information content(PIC)values indicated that the HinfⅠ-RFLP(0.25＜PIC＜0.5)were moderately polymorphic in Tibet mini-pigs, and were highly polymorphic were in other RFLPs.Conclusion:In HinfⅠ-RFLP site of 5'upstream region of H-FABP,the Tibet mini-pigs have polymorphism.We can utilize this polymorphism mark in 5'upstream region in H-FABP of Tibet mini-pig to further analyse its relation with intramuscular fat.Second part The PERV gene studies of Tibet mini-pigsBackground:On clinic,serious shortage of donor organs,make people turn sight to xenotransplantation.At present,the pig considereds to be the most suitable donor of organs in xenotransplantation.But researchers had discovered that the virus DNA integrated into in pig's somatic genome(porcine endogenous retrovirus,PERV) can infect many kinds of human cells in vitro,this has caused people's extensive concern about the xenotransplantation securities.PERV cannot be eliminated by classical mean such as SPF animal housing.The chinese domestic pigs have great differences in region distribution,evolution degree,etc.,and they have high genetic diversity,it is easy to find individuals with low or no virus copy-number,so the semi-quantitative PCR method was used in this research to estimate difference of PERV copy-number of main mini-pigs in China,we attempt to find out breeds or individuals with no or low virus copy-number,the aim of us is to cultivate pig breeds or strains that are no threaten of endogenous virus which are suitable for xenotransplantation.Methods:Collected 15 Tibet mini-pigs as the research object.Under animal's sober state,fixed it,collected 5ml of blood from anterior vena cava,EDTA was added to blood as an anticoagulant.Then kept the blood on -70℃low-temperature refrigerator.Tibet mini-pig blood genomic DNA was extracted using kit(TIANamp Genomic DNA Kit) produced by TIANGEN Company.The DNA sample of Bama mini-pigs,Guizhou mini-pigs,Wuzhishan mini-pigs were offered by professor Li Kui at Institute of Animal Sciences of Chinese Academy of Agricultural Sciences.The whole genome DNA was dissolved with TE solution,diluting to 10 times,measured OD_280nm and OD_260nm at ultraviolet spectrophotometer.Adjust DNA density to 10ng/μl according to OD_260nm value measured,kept it on -20℃low-temperature refrigerator. According to polymerase segment of PERV gene(PERVp),we designed specific primer to make the copy of PERVp represent on copy of PERV gene.Four pais of primers were designed all together to amplify PERVp,EnvA,EnvB,sexonlαrespectively(single-copy gene contrast).The length of PERVp PCR products is 326bp,the length of EnvA PCR products is 359bp,the length of EnvB PCR products is 263bp,the length of sexonlαPCR products is 388bp.Dilute to 10,5,2.5,1.25ng/μl for each DNA sample to detect single-copy contrast and EnvA;dilute to 0.625, 0.3125,0.15625 and 0.078125ng/μl further to detect PERVp and EnvB.Grey level value was estimated by Scion Image software,assume GCAT content is equal,correct grey level value to reach 300bp.EXCEL software of Microsoft was used to calculate the slope of straight line of the series dilution,the copy-number of one gene is the the ratio of slope of one gene with corresponding slope of single-copy gene.Data are expressed as(?)+s,all statistical analysis was carried out using SPSS13.0 software, comparison of mean differences between groups was done using one-way ANOVA. Differences were considered significant when P＜0.05.Results:The copy-number of PERV,EnvA,EnvB genes in Guizhou mini-pigs, Wuzhishan mini-pigs,Tibet mini-pigs,Bama mini-pigs were 34.12±16.96, 29.38±13.83,28.71±14.11,27.12±14.43(F=0.614,P=0.609),9.78±5.48,10.06±5.34,10.23±5.71,10.51±6.23(F=0.044,P=0.988),24.14±11.26,20.72±8.36, 18.35±8.50,17.60±8.65(F=1.512,P=0.221) respectively.According to these results, no significant differences were observed in the copy-number of PERV,EnvA,EnvB between four different mini-pig breeds.In several kinds of Chinese mini-pigs we investigated,all have the overlay phenomena of EnvA and EnvB,namely that the sum of EnvA and EnvB is greater than PERV gene,and this happen in many sites for some individuals.All individuals of Wuzhishan mini-pigs have the phenomenon of overlap.Conclusion:Compared to other pig strains which have been reported,four kinds of commonly used mini-pigs have relatively low endogenous virus copy-number, show its advantages used in xenotransplantation research.