盤羊雜交羊SPLUNC1基因的克
發(fā)布時間：2018-07-14 09:49
【摘要】：盤羊(♂)與巴什拜羊(♀)雜交后代具有體型大、羔羊生長發(fā)育快、瘦肉率高的特點,但該雜交羔羊易感染綿羊肺炎支原體(Mycoplasma ovipneumoniae,Mo)而死亡。短顎、肺及鼻咽上皮克隆1(short palate,lung and nasal epithelium clone 1,SPLUNC1)是新發(fā)現(xiàn)的具有宿主防御功能的生物活性物質,是宿主先天性防御肺炎支原體感染的關鍵基因。目的:本研究通過克隆盤羊雜交羊SPLUNC1基因全長c DNA序列,與盤羊及巴什拜羊SPLUNC1基因進行比較研究,并對盤羊雜交羊SPLUNC1基因進行真核表達,研究重組SPLUNC1蛋白(recombinant short palate,lung and nasal epithelium clone 1,r SPLUNC1)的抑菌作用、對體外培養(yǎng)的Mo生長的影響、對中性粒細胞彈性蛋白酶活性的影響、對呼吸道致病菌感染的淋巴細胞凋亡的影響,并與巴什拜羊r SPLUNC1蛋白的功能比較,為盤羊雜交羊r SPLUNC1的體外功能研究奠定基礎。方法:(1)盤羊雜交羊SPLUNC1基因全長c DNA序列的克隆:使用RT-PCR法和RACE法克隆盤羊雜交羊SPLUNC1基因3’端和5’端,測序后拼接獲得SPLUNC1基因全長c DNA序列。(2)盤羊雜交羊SPLUNC1基因c DNA全長序列的生物信息學分析:利用在線生物信息學分析工具及生物信息學軟件對獲得的盤羊雜交羊SPLUNC1基因全長c DNA序列進行核酸序列、編碼氨基酸以及蛋白的信號肽、亞細胞定位、高級結構及系統(tǒng)發(fā)育分析。(3)盤羊雜交羊r SPLUNC1的真核表達及純化:根據(jù)已克隆出的SPLUNC1基因全長c DNA序列設計特異性表達引物,使用RT-PCR法擴增SPLUNC1基因ORF,構建重組表達質粒p PIC9K-SPLUNC1,將重組表達質粒電擊轉化至巴斯德畢赤酵母GS115,并利用甲醇對重組菌株GS115/p PIC9K-SPLUNC1誘導表達,SDS-PAGE和Western Blot法檢測r SPLUNC1的表達;利用Ni-NTA瓊脂親和層析對真核表達的r SPLUNC1進行純化。(4)盤羊雜交羊r SPLUNC1的抑菌作用:利用微量肉湯稀釋法檢測不同濃度盤羊雜交羊r SPLUNC1對大腸桿菌、肺炎鏈球菌、巴氏桿菌及金黃色葡萄球菌的抑菌作用。(5)盤羊雜交羊r SPLUNC1對Mo生長的影響:利用平板菌落計數(shù)法和實時熒光定量PCR(FQ-PCR)法檢測盤羊雜交羊r SPLUNC1對體外培養(yǎng)的Mo生長的影響。(6)盤羊雜交羊r SPLUNC1對中性粒細胞彈性蛋白酶活性的影響:利用四肽底物法檢測r SPLUNC1對中性粒細胞彈性蛋白酶活性的影響。(7)盤羊雜交羊r SPLUNC1對致病菌感染的淋巴細胞凋亡的影響:利用DNA ladder法和Annexin V-FITC/PI雙染法檢測r SPLUNC1對致病菌感染的淋巴細胞凋亡的影響。結果與結論:(1)獲得1117bp的盤羊雜交羊SPLUNC1基因全長cDNA序列。(2)盤羊雜交羊SPLUNC1基因開放閱讀框(ORF)為768bp,編碼256個氨基酸,預測該編碼蛋白的分子量為26.57KDa,等電點為5.006。生物信息學分析表明SPLUNC1蛋白N端存在20個氨基酸殘基的信號肽,具有高疏水性,亞細胞定位在細胞外。氨基酸序列分析表明,盤羊雜交羊SPLUNC1蛋白與巴氏拜羊SPLUNC1蛋白有一處差異。(3)成功構建了盤羊雜交羊SPLUNC1的真核表達載體,并在畢赤酵母成功表達了分子量為25.96KDa的r SPLUNC1。(4)抑菌試驗結果表明,濃度在20~40μg/m L的盤羊雜交羊r SPLUNC1可以極顯著抑制(p0.01)巴氏桿菌、顯著抑制(p0.05)大腸桿菌的生長。(5)平板菌落計數(shù)結果顯示,濃度為40μg/m L的盤羊雜交羊r SPLUNC1能夠顯著(p0.05)抑制Mo在瓊脂平板上的生長。實時熒光定量PCR結果顯示,濃度為40μg/m L的盤羊雜交羊r SPLUNC1在作用4h時,Mo 16S r RNA的拷貝數(shù)與對照組相比差異極顯著(P0.01)。(6)四肽底物法結果顯示,濃度大于20μg/m L的盤羊雜交羊r SPLUNC1可以增強NE的活性,且具有劑量效應。(7)DNA ladder法和Annexin V-FITC/PI雙染法結果表明盤羊雜交羊r SPLUNC1對致病菌感染的淋巴細胞凋亡無影響。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953
，
本文編號：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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953
，
本文編號：2121265
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2121265.html
最近更新
教材專著
