中文摘要：

本研究旨在探讨通过构建同时表达大肠杆菌植酸酶appA2及人抗黏液病毒基因A（Myxovirus resistance gene,MxA）的真核表达载体,比较双基因表达载体和相应单基因表达载体中外源基因的表达效率;从pcDNA-ap-pA2载体上扩增CMV-appA2-BGHpA片段,通过MluI酶切位点连接到pcDNA-MxA载体中,构建重组载体pcDNA-appA-MxA（下称AMP）,分别将pcDNA3.1（＋）、pcDNA-MxA、pcDNA-appA2及AMP质粒转染猪PK15细胞,经G418筛选后,取细胞通过实时荧光定量PCR测定细胞内appA2及MxA的表达,同时部分细胞通过Westernblot测定细胞内MxA蛋白表达量,采用改进的钼酸铵显色法测定细胞内植酸酶活性;酶切及测序结果表明成功构建了AMP载体;QRT-PCR结果表明,转AMP细胞组MxA表达水平是转pcDNA-MxA组的1.118倍,转AMP细胞组appA2表达水平是转pcDNA-appA2组的1.134倍,上述各组间差异显著（P〈0.05）。灰度分析结果表明,AMP细胞组细胞内MxA表达量是转染pcDNA-MxA细胞组的1.07倍;植酸酶活性测定试验结果表明,转AMP、pcDNA-appA2细胞组及对照组细胞内植酸酶酶活和对照组平均酶活分别为0.294、0.235及0.082FTU·μL-1,2个试验组间差异不显著,试验组和对照组间差异极显著（P〈0.01）;试验结果表明本研究构建双基因表达载体中外源基因的表达效率比相应单基因表达载体表达效率高,本研究为以后多基因共表达及制备多基因转基因动物奠定了基础。

英文摘要：

The aims of this study were to explore the expression efficiency in double gene eukaryotic expression vector based on construction of Escherichia coli phytase gene（appA2） combined with human MxA gene Eukaryotic expression vector.CMV-appA2-BGH pA fragment was amplified by PCR from pcDNA-appA2 vector,and then ligated to the Mlu I site of the vector pcDNA-MxA.The recombinant vector pcDNA-appA-MxA（AMP）was constructed.The pcDNA3.1（＋）,pcDNA-MxA,pcDNA-appA2,and AMP vector DNA was transfected into porcine PK15 cells.Quantitative fluorogenic real-time PCR assay was performed for determining appA2 and MxA mRNA expression level.MxA protein in pcDNA3.1（＋）,pcDNA-MxA,pcDNA-appA2 and AMP cells groups was analyzed with Western blot method.The appA2 enzyme activity from pcDNA3.1（＋）,pcDNA-appA2 and AMP cells groups was dectected with modified Molybdate colorimetric method.The results of sequencing and restriction showed that the AMP vector was constructed successfully.QRT-PCR results showed that MxA mRNA expressiong level in AMP cells group was 1.118 times than that in pcDNA-MxA cells group,appA2 mRNA expression level in AMP cells group was 1.134 times than that in pcDNA-appA2 cells group,and the difference was significant（P0.05）;Results of gray intensity analysis showed that MxA protein relative expression level in AMP cells group was 1.07 times than that in pcDNA-MxA cells group.Phytase detection results showed that enzyme activity of phytase in AMP,pcDNA-appA2 and control groups were 0.294,0.235 and 0.082 FTU·μL-1,respectively,and there were no obvious difference in the two experimental groups,and the difference in the two experimental groups and control group was extremely significant（P0.01）.The results showed that the vector constructed had relatively higher expression efficiency compared with single gene expression vector.This study establish a foundation for preparation transgenic animal co-expression multiple foreign gene.