The Regulatory Effects Of Recombinant Apolipoprotein B100on Fat Metabolism In Dairy Cows

In this study, Apolipoprotein B100(ApoB100) gene was cloned and prokaryoticexpression vector of Escherichia coli (E. coli) was constructed by using the geneticengineering principle，modern cytobiology and molecular biology technique. Theexpression of ApoB100was acquired in Escherichia coli expression system. Thedetermination of biological activity of expressed product was made to demonstratethat recombinant ApoB100of E. coli expression has good biological activity. Afterthat, through the fluorescence quantitative RT-PCR methods and colorimetricmethods, and the primary culture experiments of fat cells and vitro liver cells andanimal experiments, regulatory effects of ApoB100, as the structural protein of VLDL,on the assembly and secretion of very low density lipoprotein(VLDL) and lowdensity lipoprotein (LDL) were analyzed from the cell and molecular level. This laid atheoretical foundation for the occurrence mechanism in which negative balance ofenergy metabolism in periparturient dairy cows is revealed from gene level, and alsofor the new ways in which the disease is treated and prevented from the levels ofcytokines.Cloning and sequence analysis of ApoB100was made as follows: first, totalRNA was extracted from the cow liver tissues; then primers were designed accordingto the bovine apolipoprotein B100gene sequences published by GenBank; and bovineapolipoprotein B100gene was amplified by the method of RT-PCR. The target geneacquired was inserted into pGM-T cloning vector. The pGM-T-BovApoB100genesequence which was constructed showed that the target gene was747bp, which,according to the GenBank bovine apolipoprotein gene coding sequence, shared the100%of homology. It confirmed the correction of the gene which was cloned.The expression of Apolipoprotein B100gene in Escherichia coli was made asfollows: the primer was designed and synthesized according to the requirements of E.coli expression vector pET-28a. Cloning vector pGM-T-BovApoB100was constructed. The pGM-T-BovApoB100and E. coli plasmid of pET-28a wererestricted with EcoRⅠand BamHⅠdouble enzymes to reach the construction of E.coli expression plasmid pET-28a-BovApoB100of Apolipoprotein B100gene. Therecombinant plasmids were transformed into E. coli Dh5α and the plasmid DNA wasextracted. After the DNA sequence analysis for the identification of the correctness, ithelps to obtain the successful expression of Recombinant Apolipoprotein B100.According to the cDNA sequence in dairy cows published by GenBank, primerswere designed and fragments for quantitative gene expression levels weresuccessfully amplified. The results showed that homologies of the VLDLR and LDLRNucleotide was100%. After the optimization, fluorescence quantitative RT-PCR andRT-PCR detection method with good accuracy, high sensitivity and strong stabilitywas established.The modified collagenase was used to digest calf’ fat cells and liver cells. Cellswere seeded (1×106) on the custodite cell culture dishes with polylysine and cultivatedin37℃,5%CO2culture box with liquid exchange every24hours. The cell growthwas observed by the inverted microscope. The experiment was conducted afterattachment.Fluorescence quantitative RT-PCR method was employed to determine the effectof recombinant ApoB100with different concentrations (0μg/mL、100μg/mL、200μg/mL、300μg/mL、400μg/mL) on the mRNA expression level of fat metabolismin calf’ cultured primary hepatocytes. The results demonstrated that there is a positivecorrelation between the expression of fat cells and liver cells VLDLR and LDLRmRNA and the concentration of recombinant ApoB100. There is a significantdifference between the experiment groups and control groups (P<0.05).The experiment was conducted as follows. Ten perinatal healthy cows wereselected and were divided into2groups randomly---one group with an injection ofrecombinant Apo B100and the other group with an injection of normal saline, whichwere made respectively7days before and after delivery. The expressions of VLDLRand LDLR mRNA were tested, VLDL and LDL concentration in plasma weredetermined. Results showed that there is an obvious increase in the expressions ofVLDLR and LDLR mRNA, compared with those of control group. VLDL andconcentration in plasma slightly increased in experiment group. It is concluded that, bymeans of expressions of VLDLR and LDLR mRNA, recombinant ApoB100could promote VLDL and LDL transportation so as to affect plasma lipid concentrations.