盘羊杂交羊SPLUNC1基因的克
发布时间：2018-07-14 09:49
【摘要】：盘羊(♂)与巴什拜羊(♀)杂交后代具有体型大、羔羊生长发育快、瘦肉率高的特点,但该杂交羔羊易感染绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)而死亡。短颚、肺及鼻咽上皮克隆1(short palate,lung and nasal epithelium clone 1,SPLUNC1)是新发现的具有宿主防御功能的生物活性物质,是宿主先天性防御肺炎支原体感染的关键基因。目的:本研究通过克隆盘羊杂交羊SPLUNC1基因全长c DNA序列,与盘羊及巴什拜羊SPLUNC1基因进行比较研究,并对盘羊杂交羊SPLUNC1基因进行真核表达,研究重组SPLUNC1蛋白(recombinant short palate,lung and nasal epithelium clone 1,r SPLUNC1)的抑菌作用、对体外培养的Mo生长的影响、对中性粒细胞弹性蛋白酶活性的影响、对呼吸道致病菌感染的淋巴细胞凋亡的影响,并与巴什拜羊r SPLUNC1蛋白的功能比较,为盘羊杂交羊r SPLUNC1的体外功能研究奠定基础。方法:(1)盘羊杂交羊SPLUNC1基因全长c DNA序列的克隆:使用RT-PCR法和RACE法克隆盘羊杂交羊SPLUNC1基因3’端和5’端,测序后拼接获得SPLUNC1基因全长c DNA序列。(2)盘羊杂交羊SPLUNC1基因c DNA全长序列的生物信息学分析:利用在线生物信息学分析工具及生物信息学软件对获得的盘羊杂交羊SPLUNC1基因全长c DNA序列进行核酸序列、编码氨基酸以及蛋白的信号肽、亚细胞定位、高级结构及系统发育分析。(3)盘羊杂交羊r SPLUNC1的真核表达及纯化:根据已克隆出的SPLUNC1基因全长c DNA序列设计特异性表达引物,使用RT-PCR法扩增SPLUNC1基因ORF,构建重组表达质粒p PIC9K-SPLUNC1,将重组表达质粒电击转化至巴斯德毕赤酵母GS115,并利用甲醇对重组菌株GS115/p PIC9K-SPLUNC1诱导表达,SDS-PAGE和Western Blot法检测r SPLUNC1的表达;利用Ni-NTA琼脂亲和层析对真核表达的r SPLUNC1进行纯化。(4)盘羊杂交羊r SPLUNC1的抑菌作用:利用微量肉汤稀释法检测不同浓度盘羊杂交羊r SPLUNC1对大肠杆菌、肺炎链球菌、巴氏杆菌及金黄色葡萄球菌的抑菌作用。(5)盘羊杂交羊r SPLUNC1对Mo生长的影响:利用平板菌落计数法和实时荧光定量PCR(FQ-PCR)法检测盘羊杂交羊r SPLUNC1对体外培养的Mo生长的影响。(6)盘羊杂交羊r SPLUNC1对中性粒细胞弹性蛋白酶活性的影响:利用四肽底物法检测r SPLUNC1对中性粒细胞弹性蛋白酶活性的影响。(7)盘羊杂交羊r SPLUNC1对致病菌感染的淋巴细胞凋亡的影响:利用DNA ladder法和Annexin V-FITC/PI双染法检测r SPLUNC1对致病菌感染的淋巴细胞凋亡的影响。结果与结论:(1)获得1117bp的盘羊杂交羊SPLUNC1基因全长cDNA序列。(2)盘羊杂交羊SPLUNC1基因开放阅读框(ORF)为768bp,编码256个氨基酸,预测该编码蛋白的分子量为26.57KDa,等电点为5.006。生物信息学分析表明SPLUNC1蛋白N端存在20个氨基酸残基的信号肽,具有高疏水性,亚细胞定位在细胞外。氨基酸序列分析表明,盘羊杂交羊SPLUNC1蛋白与巴氏拜羊SPLUNC1蛋白有一处差异。(3)成功构建了盘羊杂交羊SPLUNC1的真核表达载体,并在毕赤酵母成功表达了分子量为25.96KDa的r SPLUNC1。(4)抑菌试验结果表明,浓度在20~40μg/m L的盘羊杂交羊r SPLUNC1可以极显著抑制(p0.01)巴氏杆菌、显著抑制(p0.05)大肠杆菌的生长。(5)平板菌落计数结果显示,浓度为40μg/m L的盘羊杂交羊r SPLUNC1能够显著(p0.05)抑制Mo在琼脂平板上的生长。实时荧光定量PCR结果显示,浓度为40μg/m L的盘羊杂交羊r SPLUNC1在作用4h时,Mo 16S r RNA的拷贝数与对照组相比差异极显著(P0.01)。(6)四肽底物法结果显示,浓度大于20μg/m L的盘羊杂交羊r SPLUNC1可以增强NE的活性,且具有剂量效应。(7)DNA ladder法和Annexin V-FITC/PI双染法结果表明盘羊杂交羊r SPLUNC1对致病菌感染的淋巴细胞凋亡无影响。
[Abstract]:The hybrids and basbai sheep have the characteristics of large size, fast growth and high lean meat rate, but the hybrid lamb is easily infected with Mycoplasma ovipneumoniae (Mo), and the short jaw, lung and nasopharyngeal epithelial clones 1 (short palate, lung and nasal epithelium clone 1, SPLUNC1) are newly discovered The biological active substance of the host defense function is the key gene of the host's congenital defense mycoplasma infection. Objective: To compare the C DNA sequence of the SPLUNC1 gene of the sheep cross sheep and the SPLUNC1 gene of the sheep and bahash sheep, and to carry out the eukaryotic expression of the SPLUNC1 gene of the goat hybrid sheep and study the recombinant SPL. The effect of UNC1 protein (recombinant short palate, lung and nasal epithelium clone 1, R SPLUNC1) on the growth of Mo growth in vitro, the effect on the activity of neutrophil elastase, the effect on the apoptosis of lymphocytes infected by respiratory pathogenic bacteria, and compared with the function of bash sheep protein The foundation of the in vitro functional study of R SPLUNC1 was established. Methods: (1) cloning of the full length C DNA sequence of SPLUNC1 gene of sheep cross sheep: using RT-PCR and RACE to clone the SPLUNC1 gene 3 'end and 5' end of the sheep cross sheep, and sequenced the whole length C DNA sequence of the SPLUNC1 gene. (2) the whole length sequence of the sheep hybrid sheep SPLUNC1 gene. Bioinformatics analysis: using online bioinformatics analysis tools and bioinformatics software to carry out nucleic acid sequences, encoded amino acid and protein signal peptides, subcellular localization, high structure and phylogenetic analysis of SPLUNC1 gene C DNA sequence of sheep hybrid sheep. (3) eukaryotic expression and purity of R SPLUNC1 of sheep cross sheep The specific primers were designed based on the full-length C DNA sequence of the cloned SPLUNC1 gene, the SPLUNC1 gene ORF was amplified by RT-PCR, and the recombinant expression plasmid P PIC9K-SPLUNC1 was constructed. The recombinant expression plasmid was converted to the GS115 of Pichia pastoris, and the expression of the recombinant plasmid was induced by methanol to GS115/p PIC9K-SPLUNC1, SDS-PAGE and. Western Blot was used to detect the expression of R SPLUNC1, and Ni-NTA agar affinity chromatography was used to purify the eukaryotic R SPLUNC1. (4) the Bacteriostasis of R SPLUNC1 in cross sheep of pagan sheep: detection of the Bacteriostasis of Escherichia coli, Streptococcus pneumoniae, pasteurella and Staphylococcus aureus by micro broth dilution method. Effect. (5) the effect of R SPLUNC1 on the growth of Mo: the effect of R SPLUNC1 on the growth of Mo in vitro by the plate colony counting method and real-time fluorescence quantitative PCR (FQ-PCR) method. (6) the effect of R SPLUNC1 on the activity of neutrophil elastin in sheep hybrid sheep: the detection of R SPLUNC1 by four peptide substrate method The effect of neutrophil elastase activity. (7) the effect of R SPLUNC1 on the apoptosis of lymphocytes infected by pathogenic bacteria: the effect of DNA ladder and Annexin V-FITC/PI double staining on the apoptosis of lymphocytes infected by R SPLUNC1. Results and conclusions: (1) the total SPLUNC1 gene of the sheep hybrid sheep was obtained. The long cDNA sequence. (2) the SPLUNC1 gene open reading frame (ORF) of the sheep hybrid sheep (ORF) is 768bp, encodes 256 amino acids, and predicts the molecular weight of the encoded protein is 26.57KDa. The isoelectric point is 5.006. bioinformatics analysis that the SPLUNC1 protein N terminal has 20 amino acid residues, which has high hydrophobicity and subcellular localization in the extracellular. The sequence analysis showed that there was a difference between the SPLUNC1 protein of the sheep cross sheep and the barson sheep SPLUNC1 protein. (3) the eukaryotic expression vector of the sheep SPLUNC1 was successfully constructed, and the R SPLUNC1. (4) bacteriostasis test of the molecular weight of the sheep was successfully expressed in Pichia pastoris, and the concentration was in R SPLUNC1 of the sheep hybrid sheep of 20~40 u g/m L. The growth of Escherichia coli was significantly inhibited (P0.05) by inhibiting (P0.01) Pasteurella, and (5) the colony count results showed that R SPLUNC1 with a concentration of 40 mu g/m L could significantly (P0.05) inhibit the growth of Mo on the agar plate. Real time fluorescence quantitative PCR results showed that a sheep hybrid sheep R with a concentration of 40 mu g/m L was made When using 4h, the number of copies of Mo 16S R RNA was significantly different from that of the control group (P0.01). (6) the results of four peptide substrate method showed that the R SPLUNC1 of the sheep hybrid sheep with a concentration greater than 20 mu g/m L could enhance the NE activity and have a dose effect. (7) the results of both DNA enrichment and double staining showed that the infection of the sheep There was no effect on the apoptosis of lymphocyte.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953
，
本文编号：2121265
[Abstract]:The hybrids and basbai sheep have the characteristics of large size, fast growth and high lean meat rate, but the hybrid lamb is easily infected with Mycoplasma ovipneumoniae (Mo), and the short jaw, lung and nasopharyngeal epithelial clones 1 (short palate, lung and nasal epithelium clone 1, SPLUNC1) are newly discovered The biological active substance of the host defense function is the key gene of the host's congenital defense mycoplasma infection. Objective: To compare the C DNA sequence of the SPLUNC1 gene of the sheep cross sheep and the SPLUNC1 gene of the sheep and bahash sheep, and to carry out the eukaryotic expression of the SPLUNC1 gene of the goat hybrid sheep and study the recombinant SPL. The effect of UNC1 protein (recombinant short palate, lung and nasal epithelium clone 1, R SPLUNC1) on the growth of Mo growth in vitro, the effect on the activity of neutrophil elastase, the effect on the apoptosis of lymphocytes infected by respiratory pathogenic bacteria, and compared with the function of bash sheep protein The foundation of the in vitro functional study of R SPLUNC1 was established. Methods: (1) cloning of the full length C DNA sequence of SPLUNC1 gene of sheep cross sheep: using RT-PCR and RACE to clone the SPLUNC1 gene 3 'end and 5' end of the sheep cross sheep, and sequenced the whole length C DNA sequence of the SPLUNC1 gene. (2) the whole length sequence of the sheep hybrid sheep SPLUNC1 gene. Bioinformatics analysis: using online bioinformatics analysis tools and bioinformatics software to carry out nucleic acid sequences, encoded amino acid and protein signal peptides, subcellular localization, high structure and phylogenetic analysis of SPLUNC1 gene C DNA sequence of sheep hybrid sheep. (3) eukaryotic expression and purity of R SPLUNC1 of sheep cross sheep The specific primers were designed based on the full-length C DNA sequence of the cloned SPLUNC1 gene, the SPLUNC1 gene ORF was amplified by RT-PCR, and the recombinant expression plasmid P PIC9K-SPLUNC1 was constructed. The recombinant expression plasmid was converted to the GS115 of Pichia pastoris, and the expression of the recombinant plasmid was induced by methanol to GS115/p PIC9K-SPLUNC1, SDS-PAGE and. Western Blot was used to detect the expression of R SPLUNC1, and Ni-NTA agar affinity chromatography was used to purify the eukaryotic R SPLUNC1. (4) the Bacteriostasis of R SPLUNC1 in cross sheep of pagan sheep: detection of the Bacteriostasis of Escherichia coli, Streptococcus pneumoniae, pasteurella and Staphylococcus aureus by micro broth dilution method. Effect. (5) the effect of R SPLUNC1 on the growth of Mo: the effect of R SPLUNC1 on the growth of Mo in vitro by the plate colony counting method and real-time fluorescence quantitative PCR (FQ-PCR) method. (6) the effect of R SPLUNC1 on the activity of neutrophil elastin in sheep hybrid sheep: the detection of R SPLUNC1 by four peptide substrate method The effect of neutrophil elastase activity. (7) the effect of R SPLUNC1 on the apoptosis of lymphocytes infected by pathogenic bacteria: the effect of DNA ladder and Annexin V-FITC/PI double staining on the apoptosis of lymphocytes infected by R SPLUNC1. Results and conclusions: (1) the total SPLUNC1 gene of the sheep hybrid sheep was obtained. The long cDNA sequence. (2) the SPLUNC1 gene open reading frame (ORF) of the sheep hybrid sheep (ORF) is 768bp, encodes 256 amino acids, and predicts the molecular weight of the encoded protein is 26.57KDa. The isoelectric point is 5.006. bioinformatics analysis that the SPLUNC1 protein N terminal has 20 amino acid residues, which has high hydrophobicity and subcellular localization in the extracellular. The sequence analysis showed that there was a difference between the SPLUNC1 protein of the sheep cross sheep and the barson sheep SPLUNC1 protein. (3) the eukaryotic expression vector of the sheep SPLUNC1 was successfully constructed, and the R SPLUNC1. (4) bacteriostasis test of the molecular weight of the sheep was successfully expressed in Pichia pastoris, and the concentration was in R SPLUNC1 of the sheep hybrid sheep of 20~40 u g/m L. The growth of Escherichia coli was significantly inhibited (P0.05) by inhibiting (P0.01) Pasteurella, and (5) the colony count results showed that R SPLUNC1 with a concentration of 40 mu g/m L could significantly (P0.05) inhibit the growth of Mo on the agar plate. Real time fluorescence quantitative PCR results showed that a sheep hybrid sheep R with a concentration of 40 mu g/m L was made When using 4h, the number of copies of Mo 16S R RNA was significantly different from that of the control group (P0.01). (6) the results of four peptide substrate method showed that the R SPLUNC1 of the sheep hybrid sheep with a concentration greater than 20 mu g/m L could enhance the NE activity and have a dose effect. (7) the results of both DNA enrichment and double staining showed that the infection of the sheep There was no effect on the apoptosis of lymphocyte.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953
，
本文编号：2121265
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