盤(pán)羊雜交羊SPLUNC1基因的克
發(fā)布時(shí)間：2018-07-14 09:49
【摘要】：盤(pán)羊(♂)與巴什拜羊(♀)雜交后代具有體型大、羔羊生長(zhǎng)發(fā)育快、瘦肉率高的特點(diǎn),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(zhì),是宿主先天性防御肺炎支原體感染的關(guān)鍵基因。目的:本研究通過(guò)克隆盤(pán)羊雜交羊SPLUNC1基因全長(zhǎng)c DNA序列,與盤(pán)羊及巴什拜羊SPLUNC1基因進(jìn)行比較研究,并對(duì)盤(pán)羊雜交羊SPLUNC1基因進(jìn)行真核表達(dá),研究重組SPLUNC1蛋白(recombinant short palate,lung and nasal epithelium clone 1,r SPLUNC1)的抑菌作用、對(duì)體外培養(yǎng)的Mo生長(zhǎng)的影響、對(duì)中性粒細(xì)胞彈性蛋白酶活性的影響、對(duì)呼吸道致病菌感染的淋巴細(xì)胞凋亡的影響,并與巴什拜羊r SPLUNC1蛋白的功能比較,為盤(pán)羊雜交羊r SPLUNC1的體外功能研究奠定基礎(chǔ)。方法:(1)盤(pán)羊雜交羊SPLUNC1基因全長(zhǎng)c DNA序列的克隆:使用RT-PCR法和RACE法克隆盤(pán)羊雜交羊SPLUNC1基因3’端和5’端,測(cè)序后拼接獲得SPLUNC1基因全長(zhǎng)c DNA序列。(2)盤(pán)羊雜交羊SPLUNC1基因c DNA全長(zhǎng)序列的生物信息學(xué)分析:利用在線生物信息學(xué)分析工具及生物信息學(xué)軟件對(duì)獲得的盤(pán)羊雜交羊SPLUNC1基因全長(zhǎng)c DNA序列進(jìn)行核酸序列、編碼氨基酸以及蛋白的信號(hào)肽、亞細(xì)胞定位、高級(jí)結(jié)構(gòu)及系統(tǒng)發(fā)育分析。(3)盤(pán)羊雜交羊r SPLUNC1的真核表達(dá)及純化:根據(jù)已克隆出的SPLUNC1基因全長(zhǎng)c DNA序列設(shè)計(jì)特異性表達(dá)引物,使用RT-PCR法擴(kuò)增SPLUNC1基因ORF,構(gòu)建重組表達(dá)質(zhì)粒p PIC9K-SPLUNC1,將重組表達(dá)質(zhì)粒電擊轉(zhuǎn)化至巴斯德畢赤酵母GS115,并利用甲醇對(duì)重組菌株GS115/p PIC9K-SPLUNC1誘導(dǎo)表達(dá),SDS-PAGE和Western Blot法檢測(cè)r SPLUNC1的表達(dá);利用Ni-NTA瓊脂親和層析對(duì)真核表達(dá)的r SPLUNC1進(jìn)行純化。(4)盤(pán)羊雜交羊r SPLUNC1的抑菌作用:利用微量肉湯稀釋法檢測(cè)不同濃度盤(pán)羊雜交羊r SPLUNC1對(duì)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(pán)羊雜交羊r SPLUNC1對(duì)Mo生長(zhǎng)的影響:利用平板菌落計(jì)數(shù)法和實(shí)時(shí)熒光定量PCR(FQ-PCR)法檢測(cè)盤(pán)羊雜交羊r SPLUNC1對(duì)體外培養(yǎng)的Mo生長(zhǎng)的影響。(6)盤(pán)羊雜交羊r SPLUNC1對(duì)中性粒細(xì)胞彈性蛋白酶活性的影響:利用四肽底物法檢測(cè)r SPLUNC1對(duì)中性粒細(xì)胞彈性蛋白酶活性的影響。(7)盤(pán)羊雜交羊r SPLUNC1對(duì)致病菌感染的淋巴細(xì)胞凋亡的影響:利用DNA ladder法和Annexin V-FITC/PI雙染法檢測(cè)r SPLUNC1對(duì)致病菌感染的淋巴細(xì)胞凋亡的影響。結(jié)果與結(jié)論:(1)獲得1117bp的盤(pán)羊雜交羊SPLUNC1基因全長(zhǎng)cDNA序列。(2)盤(pán)羊雜交羊SPLUNC1基因開(kāi)放閱讀框(ORF)為768bp,編碼256個(gè)氨基酸,預(yù)測(cè)該編碼蛋白的分子量為26.57KDa,等電點(diǎn)為5.006。生物信息學(xué)分析表明SPLUNC1蛋白N端存在20個(gè)氨基酸殘基的信號(hào)肽,具有高疏水性,亞細(xì)胞定位在細(xì)胞外。氨基酸序列分析表明,盤(pán)羊雜交羊SPLUNC1蛋白與巴氏拜羊SPLUNC1蛋白有一處差異。(3)成功構(gòu)建了盤(pán)羊雜交羊SPLUNC1的真核表達(dá)載體,并在畢赤酵母成功表達(dá)了分子量為25.96KDa的r SPLUNC1。(4)抑菌試驗(yàn)結(jié)果表明,濃度在20~40μg/m L的盤(pán)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(zhǎng)。(5)平板菌落計(jì)數(shù)結(jié)果顯示,濃度為40μg/m L的盤(pán)羊雜交羊r SPLUNC1能夠顯著(p0.05)抑制Mo在瓊脂平板上的生長(zhǎng)。實(shí)時(shí)熒光定量PCR結(jié)果顯示,濃度為40μg/m L的盤(pán)羊雜交羊r SPLUNC1在作用4h時(shí),Mo 16S r RNA的拷貝數(shù)與對(duì)照組相比差異極顯著(P0.01)。(6)四肽底物法結(jié)果顯示,濃度大于20μg/m L的盤(pán)羊雜交羊r SPLUNC1可以增強(qiáng)NE的活性,且具有劑量效應(yīng)。(7)DNA ladder法和Annexin V-FITC/PI雙染法結(jié)果表明盤(pán)羊雜交羊r SPLUNC1對(duì)致病菌感染的淋巴細(xì)胞凋亡無(wú)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(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q953
，
本文編號(hào)：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(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q953
，
本文編號(hào)：2121265
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