抗菌肽DYBOWSKIN-2CDYA的克隆表达及其在家禽饲料中的应用

发布时间：2018-05-05 01:02

本文选题：东北林蛙抗菌肽 + cDNA克隆　；参考：《沈阳农业大学》2015年博士论文

【摘要】：本研究根据多种蛙类抗菌肽基因的5'端保守区域设计简并引物,并利用该引物对东北林蛙皮肤cDNA文库进行扩增,得到东北林蛙抗菌肽基因文库并对其进行序列测定。用CLUSTALW软件对测序结果采进行对比,结果显示:得到的11种东北林蛙抗菌肽基因中,属于Temporin家族的有dybowskin-5、dybowskin-6、dybowsin-7、dybowskin-8和dybowskin-9;属于 Brevinin-1抗菌肽家族的有dybowskin-1、dybowskin-2、dybowskin-3;抗菌肽dybowskin-4与日本林蛙抗菌肽Japonicin-1序列基本一致,14个氨基酸残基中仅相差一个。抗菌肽dybowskin-10和dybowskin-11与以往发现的其它蛙类抗菌肽序列有很小的同源性,为本实验室首次发现并命名的天然新型林蛙皮抗菌肽,属于新报道的抗菌肽家族,分别命名为:Dybowskin-2CDYa(序列为:SAVGRHGRRFGLRKHRKH)、Dybowskin-2CDYb(序列为:SAVGRHSRRFGLRKHRKH)。通过 EXPASY 生物软件对抗菌肽 Dybowskin-2CDYa、Dybowskin-2CDYb进行理化性质预测,其理化性质与其它抗菌肽性质有明显差别:富含Arg,带有较多正电荷;C末端不进行酰胺化修饰;等电点较高,为强碱性多肽。将Dybowskin-2CDYa基因与毕赤酵母分泌型表达载体pPICZα连接,构建pPICZα/Dybowskin-2CDYa表达载体,DNA测序结果显示重组质粒读码框与设计完全一致。将该表达质粒转化入毕赤酵母X33中进行甲醇诱导表达。发酵液经离心、三氯乙酸沉淀后进行电泳分析。15%Tricine-SDS-PAGE结果显示:在蛋白标准品2kDa位置处出现目的条带。对发酵上清液进行反相色谱分析,收集主要的色谱洗脱峰,分别冻干后进行抗菌活性检测。结果显示保留时间为21.78 min的洗脱峰具有抗菌活性。其峰面积占总峰面积6.32%。发酵上清液中蛋白含量为4.79 μg/μL。面积归一法计算融合多肽的理论表达量约为0.30 g/L。利用半制备反向色谱柱(10×150 mm,5 μm,ODS)对发酵液进行纯化。琼脂扩散法检测纯化后Dybowskin-2CDYa的抑菌活性。结果显示:其对蜡状芽孢杆菌、大肠杆菌、溶血鲍曼不动杆菌、产气肠杆菌等多种受试菌株均有抑菌活性。对Dybowskin-2CDYa进行生物信息学分析,结果显示其为不稳定性结构。重组Dybowskin-2CDYa的热稳定性实验证实,其在100℃加热5min即可失去抑菌活性。本研究将Dybowskin-2CDYa序列中全部的Arg替换为Lys,并命名为RK6(SAVGKHGKKFGLKKHKKH)。热稳定性试验证明RK6的稳定性优于Dybowskin-2CDYa。抗菌试验证明改造后的RK6,抗菌活性不受影响。利用SOE-PCR方法获得RK6基因并连接酵母分泌型表达载体pPICZα-A,构建pPICZα-RK6重组表达质粒,利用限制性内切酶SacI对重组质粒经酶切线性化并利用电转化方法转化巴斯德毕赤酵母X33。利用Zeocin筛选阳性转化株,经PCR方法验证后进行甲醇诱导表达。对发酵液上清进行抑菌效果测定。结果显示:重组抗菌肽RK6对革兰氏阴性菌(大肠杆菌E.coli)、革兰氏阳性菌(金黄色葡萄球菌S.au)均有抑制活性。本研究进一步构建了枯草芽孢杆菌表达系统,将RK6基因的上游连接枯草芽孢杆菌蔗糖果聚糖酶基因(SacB)的启动子-信号肽序列(SacR)、检测标签GST-Tag,得到融合序列GSR。将融合序列GSR与枯草芽孢杆菌表达载体pHY300PLK连接,构建表达质粒pHY-GSR。转化枯草芽孢杆菌WB600并利用蔗糖进行诱导。结果显示:抗菌肽RK6被成功表达并具备抗菌活性。发酵液添加饲料中对雏鸡进行饲喂。动物实验结果显示:饲料中添加发酵液可提高了雏鸡对饲料的利用率、降低雏鸡生长的料肉比;雏鸡的免疫器官相对重量及血清中部分免疫指标(γ-IFN、sIgA、IL-2、LZM)有显著提高。本文研究结论:1.从东北林蛙抗菌肽cDNA文库中筛选得到的11种抗菌肽基因中Dybowskin-2CDYa、Dybowskin-2CDYb 为抗菌肽新家族序列;2.通过抑菌试验证实:Dybowskin-2CDYα对大肠杆菌、金黄色葡糖球菌等多种受试菌均有明显的抑菌活性;3.将Dybowskin-2CDYα通过氨基酸替换改造成RK6,消除其不稳定性,改造后的RK6具有抗菌活性同时热稳定性增加;4.枯草芽孢杆菌表达系统可以对RK6进行表达。发酵液应用于饲料中可以提高饲喂动物的饲料利用率,并对实验动物的免疫器官相对重量及血清免疫指标水平有明显的提高。
[Abstract]:This study was based on the design of degenerate primers in the 5'terminal conserved region of a variety of frog antimicrobial peptides. The primers were used to amplify the cDNA Library of the northeast forest frog skin. The DNA library of the northeast forest frog's antibacterial peptide gene was obtained and the sequence was measured. The results were compared with the results of CLUSTALW software. The results showed that 11 kinds of northeast forest frogs were obtained. In the antimicrobial peptide gene, the Temporin family belongs to dybowskin-5, dybowskin-6, dybowsin-7, dybowskin-8 and dybowskin-9; it belongs to the Brevinin-1 antibacterial peptide family with dybowskin-1, dybowskin-2, dybowskin-3; the antibacterial peptide dybowskin-4 is in accordance with the Japanese Rana antiseptic peptide Japonicin-1 sequence, only one of the 14 amino acid residues is different. Peptide dybowskin-10 and dybowskin-11 have very small homology with other breast antiseptic peptide sequences found in the past. The natural new forest frog skin antibacterial peptide, which is first discovered and named in our laboratory, belongs to the newly reported antibacterial peptide family, named Dybowskin-2CDYa (sequence: SAVGRHGRRFGLRKHRKH) and Dybowskin-2CDYb (sequence: SAVGRHSRRF). GLRKHRKH). The physical and chemical properties of Dybowskin-2CDYb were predicted by EXPASY biosoftware against bacterial peptide Dybowskin-2CDYa, and their physical and chemical properties were significantly different from other antimicrobial peptides: rich in Arg, with more positive charges, no amidation of the C terminal and high isoelectric point, as a strong alkaline polypeptide. Dybowskin-2CDYa gene and Pichia pastoris The secretory expression vector pPICZ alpha was connected to construct the expression vector of pPICZ alpha /Dybowskin-2CDYa. DNA sequencing results showed that the recombinant plasmid read code frame was identical with the design. The expression plasmid was transformed into Pichia pastoris X33 for methanol induction. The fermentation liquid was centrifuged and three chloroacetic acid was precipitated by electrophoretic analysis of.15%Tricine-SDS-PAGE results. The target bands appeared at the position of the protein standard 2kDa. The fermentation supernatant was analyzed by reversed phase chromatography, the main chromatographic peaks were collected, and the antibacterial activity was detected after the freeze drying. The results showed that the peak area of the elution peak with the retention time of 21.78 min was antibacterial activity. The peak area accounted for the protein content in the 6.32%. fermentation supernatant. The theoretical expression of fusion peptide was calculated for 4.79 mu g/ mu L. area normalization. The fermentation broth was purified by half preparation of reverse chromatography column (10 x 150 mm, 5 mu m, ODS). The antimicrobial activity of Dybowskin-2CDYa was detected by agar diffusion method. The results showed that it was produced by Bacillus cereus, Escherichia coli, and Acinetobacter Bauman. A variety of strains of Enterobacteriaceae had bacteriostasis. The bioinformatics analysis of Dybowskin-2CDYa showed that it was an unstable structure. The thermal stability test of the recombinant Dybowskin-2CDYa proved that it could lose the bacteriostasis activity at 100 C for 5min. This study replaced all Arg in the Dybowskin-2CDYa sequence as Lys. RK6 (SAVGKHGKKFGLKKHKKH). The thermal stability test showed that the stability of RK6 was better than that of the Dybowskin-2CDYa. antibacterial test. The antibacterial activity was not affected. The SOE-PCR method was used to obtain the RK6 gene and to connect the yeast secretory expression vector pPICZ a -A, to construct the pPICZ alpha -RK6 recombinant expression plasmid, and to use the restriction endonuclease SacI pairs. The recombinant plasmids were transformed by enzyme tangent and converted to X33. X33. using Zeocin to screen positive transformants by using PCR method. The results showed that the recombinant antibacterial peptide RK6 was positive for Gram-negative bacteria (Escherichia coli E.coli) and Gram-positive bacteria. (Staphylococcus aureus S.au) had inhibitory activity. This study further constructed a Bacillus subtilis expression system. The upstream of the RK6 gene was connected to the promoter of the sugarcane candy sugar glycan gene (SacB) of Bacillus subtilis (SacR), and the label GST-Tag was detected. The fusion sequence GSR. was obtained for the fusion sequence GSR and Bacillus subtilis. The expression plasmid pHY-GSR. was constructed to convert Bacillus subtilis WB600 and induced by sucrose. The results showed that the antibacterial peptide RK6 was successfully expressed and had antibacterial activity. The broiler was fed in the feed of the fermented liquid. The results of animal experiment showed that adding fermentation broth in the feed could improve the benefit of chicken feed to feed. The ratio of use rate to reduce the ratio of meat and meat to the growth of chicks; the relative weight of immune organs and some immune indexes in serum (gamma -IFN, sIgA, IL-2, LZM) of chicks improved significantly. The conclusion of this paper: 1. the 11 kinds of antimicrobial peptide genes obtained from the anti antimicrobial peptide cDNA Library of the northeast forest frog are Dybowskin-2CDYa, Dybowskin-2CDYb is a new family of antibacterial peptide family; 2. The bacteriostasis test confirmed that Dybowskin-2CDY alpha had obvious bacteriostasis activity to Escherichia coli, Staphylococcus aureus and other bacteria. 3. Dybowskin-2CDY a was replaced by amino acid and transformed into RK6 to eliminate its instability. The transformed RK6 had antibacterial activity and increased thermal stability; 4. Bacillus subtilis expression system could be used. The expression of RK6. The fermentation broth should be used in feed to improve the feed utilization of feed animals, and the relative weight of immune organs and the level of serum immune indexes of experimental animals are obviously improved.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q78;S816

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