蒙古绵羊β-防御素-1（sBD-1）基因真核表达载体的构建及其在牛胎儿成纤维细胞中的表达
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摘要
β-防御素主要分布在脊椎动物的皮肤、黏膜等上皮组织,是防御素家族中的的重要成员,构成机体抵御微生物侵袭的第一道化学屏障,已被确认为是黏膜表面抗微生物屏障的组成成分。
为利用生物工程技术大量制备抗菌活性高、细胞毒性低的β-防御素,本研究以实验室保存质粒pMD19-TSinple-sBD-1为模板,利用合成的特异性引物,通过PCR方法扩增sBD-1基因,并将sBD-1基因重组到带有增强型绿色荧光蛋白的真核表达载体pEGFP-C1中,进行PCR鉴定。同时,采用脂质体转染法将构建正确的pEGFP-C1- sBD-1重组质粒转染牛胎儿成纤维细胞。转染成功后,应用实时荧光定量PCR技术检测sBD-1基因在牛胎儿成纤维细胞中的表达情况。
结果显示,经PCR扩增获得了300bp左右的产物。重组质粒载体经PCR鉴定和DNA序列测定,克隆的蒙古绵羊sBD-1基因序列与已发表序列的同源性为100 %,证明该载体构建完全正确,可以进行转染。转染细胞培养24h后,在荧光显微镜下可见大量的绿色荧光,证明构建的sBD-1基因表达载体能够在真核细胞中表达。应用实时荧光定量PCR技术进行检测,结果显示sBD-1基因在牛胎儿成纤维细胞中得以表达,这为利用基因工程大规模生产防御素奠定了基础。
β-defensin is an important members of the family and is mainly expressed in the skin, mucous membranes and other epithelial tissue of vertebrate, constitute the body against microbial invasion of the first chemical barrier to mucosal surfaces has been recognized as an integral anti-microbial barrier components.
In order to preparation A lot of antibacterial activity, low toxicity ofβ-defensin by biological engineering technology, the recombinant plasmid PMD19- TSinple- sBD-1 which was preserved in our laboratory with target sBD-1 gene was amplified by specific primers. The purified PCR product was cloned in a eukaryotic expression vector pEGFP-C1 with the enhanced green fluorescent protein. The recombined eukaryotic expression vector, pEGFP-C1-sBD-1, was transfected into bovine fetal fibroblast cells mediated by Liposome.The expression of sBD-1 in bovine fetal fibroblast cells was examined by real time RT-PCR
The result shows that the target sBD-1 gene was obtained. After the PCR analysis of recombinant plasmid and the result of cDNA sequencing demonstrated that with the homology is 100%.These results show that the vector completely correct and can be transfected. After 24h, we observed by fluorescence microscope with a large number of green fluorescence, proved sBD-1 gene can be expressed in eukaryotic cells. Application of real-time fluorescence quantitative PCR detection showed that sBD-1 gene in the bovine fetal fibroblast cells can be highly expressed. To further study the sheepβ-defensin function and the vector of animal mammary gland bioreactor used in the research foundation. To further study the sheepβ-defensin function and the vector of animal mammary gland bioreactor used in the research foundation.
为利用生物工程技术大量制备抗菌活性高、细胞毒性低的β-防御素,本研究以实验室保存质粒pMD19-TSinple-sBD-1为模板,利用合成的特异性引物,通过PCR方法扩增sBD-1基因,并将sBD-1基因重组到带有增强型绿色荧光蛋白的真核表达载体pEGFP-C1中,进行PCR鉴定。同时,采用脂质体转染法将构建正确的pEGFP-C1- sBD-1重组质粒转染牛胎儿成纤维细胞。转染成功后,应用实时荧光定量PCR技术检测sBD-1基因在牛胎儿成纤维细胞中的表达情况。
结果显示,经PCR扩增获得了300bp左右的产物。重组质粒载体经PCR鉴定和DNA序列测定,克隆的蒙古绵羊sBD-1基因序列与已发表序列的同源性为100 %,证明该载体构建完全正确,可以进行转染。转染细胞培养24h后,在荧光显微镜下可见大量的绿色荧光,证明构建的sBD-1基因表达载体能够在真核细胞中表达。应用实时荧光定量PCR技术进行检测,结果显示sBD-1基因在牛胎儿成纤维细胞中得以表达,这为利用基因工程大规模生产防御素奠定了基础。
β-defensin is an important members of the family and is mainly expressed in the skin, mucous membranes and other epithelial tissue of vertebrate, constitute the body against microbial invasion of the first chemical barrier to mucosal surfaces has been recognized as an integral anti-microbial barrier components.
In order to preparation A lot of antibacterial activity, low toxicity ofβ-defensin by biological engineering technology, the recombinant plasmid PMD19- TSinple- sBD-1 which was preserved in our laboratory with target sBD-1 gene was amplified by specific primers. The purified PCR product was cloned in a eukaryotic expression vector pEGFP-C1 with the enhanced green fluorescent protein. The recombined eukaryotic expression vector, pEGFP-C1-sBD-1, was transfected into bovine fetal fibroblast cells mediated by Liposome.The expression of sBD-1 in bovine fetal fibroblast cells was examined by real time RT-PCR
The result shows that the target sBD-1 gene was obtained. After the PCR analysis of recombinant plasmid and the result of cDNA sequencing demonstrated that with the homology is 100%.These results show that the vector completely correct and can be transfected. After 24h, we observed by fluorescence microscope with a large number of green fluorescence, proved sBD-1 gene can be expressed in eukaryotic cells. Application of real-time fluorescence quantitative PCR detection showed that sBD-1 gene in the bovine fetal fibroblast cells can be highly expressed. To further study the sheepβ-defensin function and the vector of animal mammary gland bioreactor used in the research foundation. To further study the sheepβ-defensin function and the vector of animal mammary gland bioreactor used in the research foundation.
引文
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2李洪军,胡宗利,魏泓,防御素(defensins)研究进展[J].生物工程进展,2001,20(3)34-37
3 Hao H N,Zhao J,Lotoczky G,et al.Induction of humanβ-defensin-2 in human astrocytes by lipopolysaccharide and cytokines[J].J Neurochem,2001,77 (4): 1027- 1035
4 Chae S W,Lee S H,Cho J H,et al.Expression of humanβ-defensin 1 mRNA in human palatine tonsil[J].Acta Otolaryngol,2001,121(3):414-418
5 Sahasrabudhe KS,Kimball JR,Morton TH,et al.Expression of the antimicrobial peptide, human beta-defensin 1,in duct cells of minor salivary glands and detection in saliva[J].J Dent Res,2000,79(9):1669-1674
6 Liu L,Ganz T.The proregion of human neutrophil defensin contains a motif that is essential for normal subcellular sorting[J].Blood,1995,85(4):1095-1103
7 Cornet B,Bonmatin J M,Hertru C,et al.Refined three-dimensional solution structure of insect defensin A[J].Structure,1995,3(5):435-448.
8 DIAMOND G,ZASLOFF M,ECK H, et al. Tracheal antimicrobial peptide, a cysteine rich peptide from mammalian tracheal nucosa: peptide isolation and cloning of cDNA[J].Pro NatlAcad SciUSA,1991,88(9):3952-3956.
9 RADHAKRISHNAN Y, HAMIL K G, YENUGUET S, et al. Identification, characterization and evolution of a primateβ-defens gene cluster[J]. Genes and Immunity,2005,6: 203-210.
10高真贞,董开忠.人防御素研究进展[J].西北民族大学学报,2007,28(66):62-66.
11 BONIOTTO M,TOSSIA,DELPERO M,et al.Evolution of the beta defensin 2 gene in primates[J].Genes and Immunity,2003(4):251-257.
12 KLBVER E,ADERMANN K,SCHULZ A.Synthesis and structure-activity relationship ofβ-defensins, multi-functional peptides of the immune system[J]. Journal of Peptide Science, 2006,12(4):243-357.
13 KRISHNAKUMARI V, SHARADADEVI A, SINGH S, et al. Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovineβ-defensin-2: Effects of introducing theβ-hairpin nucleating sequence D-pro-gly on antibacterial activity and biophysical properties[J].Biochemistry, 2003,42:9307–9315.
14 Jia H P, Schutte B C, Schudy A, et al. Discovery of new humanβ-defensins using a genomics-based approach [J].Gene,2001,263(1-2):211-218.
15王静华,李有志,汪以真.哺乳动物体内的防御素[J].中国兽医杂志,2004,40(1):45-47.
16 Liu L,Zhao C,Heng H H Q,et al. The Human beta-Defensin-1 and alpha-Defensins Are Encoded by Adjacent Genes: Two Peptide Families with Differing Disulfide Topology Share a Common Ancestry[J].Genomics,1997,43(3):316-320.
17 Bevins C L,Jones D E,Dutra A,et al.Human enteric defensin genes:chromosomal map position and a model for possible evolutionary relationships[J].Genomics,1996, 31(1):95-106.
18 Ouellette A J,Pravtcheva D,Ruddle F H,et al.Localization of the cryptdin locus on mouse chromosome 8[J].Genomics,1989,5(2):233-239.
19 Iannuzzi L,Gallagher D S,Di Meo G P,et al.High-resolution FISH mapping of beta-defensin genes to river buffalo and sheep chromosomes suggests a chromosome discrepancy in ffttle standard karyotypes.Cytogenet Cell Genet,1996,75:10-13.
20 Gallagher D S,Ryan A M,Diamond G,et al.Somatic cell mapping ofβ-defensin genesto cattle syntenic group U25 and fluorescent in situ hybridization localization to chromosome [J].Mamm Genome, 1995,6(8):554-556.
21 Hughes A L.Evolutionary diversification of the mammalian defensins[J].Cell Mol Life Sci, 1999,56(12):94-103.
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