摘要
本研究利用现代临床检验技术及分子生物学实验技术，对围产期高产和低产奶牛的部分血液生化指标进行了比较分析，初步探讨了高产健康奶牛与酮病及亚临床酮病对奶牛产量的影响，研究了在不同情况下与围产期酮病、脂肪肝发生密切相关的载脂蛋白B100在血液中的含量。克隆了牛载脂蛋白B100基因，构建了载脂蛋白B100表达载体，进行了大肠杆菌的原核表达；并且证明了表达的重组载脂蛋白B100具有较好的生物学活性。应用荧光定量RT-PCR方法，应用所表达的重组载脂蛋白B100，进行了动物实验，研究了载脂蛋白B100对脂肪代谢关键受体VLDLR和LDLR mRNA表达水平调控作用，为从细胞因子水平寻找围产期奶牛能量代谢负平衡的发生机制，以及应用现代生物学手段防治该类疾病的新方法进行了实验探索。
载脂蛋白B100基因的克隆与序列分析表明，我们获得的747bp目的基因与GenBank中牛载脂蛋白B100基因的编码序列相比较，同源性为100%，证明所克隆的基因序列是完全正确的。
大肠杆菌原核表达实验表明，载脂蛋白B100基因被正确整合到了受体菌GS115的基因组上，牛重组载脂蛋白B100成功实现分泌表达。
建立了VLDLR和LDLR mRNA荧光定量RT-PCR方法，荧光定量RT-PCR方法灵敏度高、准确性好、稳定性强，初步应用于VLDLR和LDLR mRNA表达水平的定量分析。
动物实验表明载脂蛋白B100对脂肪细胞和肝细胞VLDLR和LDLR mRNA的表达具有显著的上调作用。ApoB100通过对VLDLR和LDL RmRNA表达的上调来促进肝脏脂肪代谢产物VLDL和LDL从肝脏向外周组织转运，从而降低肝脏脂肪沉积。
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.
引文
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