本文選題：東北林蛙抗菌肽 + cDNA克隆��； 參考：《沈陽農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：本研究根據(jù)多種蛙類抗菌肽基因的5'端保守區(qū)域設(shè)計簡并引物,并利用該引物對東北林蛙皮膚cDNA文庫進(jìn)行擴(kuò)增,得到東北林蛙抗菌肽基因文庫并對其進(jìn)行序列測定。用CLUSTALW軟件對測序結(jié)果采進(jìn)行對比,結(jié)果顯示:得到的11種東北林蛙抗菌肽基因中,屬于Temporin家族的有dybowskin-5、dybowskin-6、dybowsin-7、dybowskin-8和dybowskin-9;屬于 Brevinin-1抗菌肽家族的有dybowskin-1、dybowskin-2、dybowskin-3;抗菌肽dybowskin-4與日本林蛙抗菌肽Japonicin-1序列基本一致,14個氨基酸殘基中僅相差一個�？咕膁ybowskin-10和dybowskin-11與以往發(fā)現(xiàn)的其它蛙類抗菌肽序列有很小的同源性,為本實(shí)驗(yàn)室首次發(fā)現(xiàn)并命名的天然新型林蛙皮抗菌肽,屬于新報道的抗菌肽家族,分別命名為:Dybowskin-2CDYa(序列為:SAVGRHGRRFGLRKHRKH)、Dybowskin-2CDYb(序列為:SAVGRHSRRFGLRKHRKH)。通過 EXPASY 生物軟件對抗菌肽 Dybowskin-2CDYa、Dybowskin-2CDYb進(jìn)行理化性質(zhì)預(yù)測,其理化性質(zhì)與其它抗菌肽性質(zhì)有明顯差別:富含Arg,帶有較多正電荷;C末端不進(jìn)行酰胺化修飾;等電點(diǎn)較高,為強(qiáng)堿性多肽。將Dybowskin-2CDYa基因與畢赤酵母分泌型表達(dá)載體pPICZα連接,構(gòu)建pPICZα/Dybowskin-2CDYa表達(dá)載體,DNA測序結(jié)果顯示重組質(zhì)粒讀碼框與設(shè)計完全一致。將該表達(dá)質(zhì)粒轉(zhuǎn)化入畢赤酵母X33中進(jìn)行甲醇誘導(dǎo)表達(dá)。發(fā)酵液經(jīng)離心、三氯乙酸沉淀后進(jìn)行電泳分析。15%Tricine-SDS-PAGE結(jié)果顯示:在蛋白標(biāo)準(zhǔn)品2kDa位置處出現(xiàn)目的條帶。對發(fā)酵上清液進(jìn)行反相色譜分析,收集主要的色譜洗脫峰,分別凍干后進(jìn)行抗菌活性檢測。結(jié)果顯示保留時間為21.78 min的洗脫峰具有抗菌活性。其峰面積占總峰面積6.32%。發(fā)酵上清液中蛋白含量為4.79 μg/μL。面積歸一法計算融合多肽的理論表達(dá)量約為0.30 g/L。利用半制備反向色譜柱(10×150 mm,5 μm,ODS)對發(fā)酵液進(jìn)行純化。瓊脂擴(kuò)散法檢測純化后Dybowskin-2CDYa的抑菌活性。結(jié)果顯示:其對蠟狀芽孢桿菌、大腸桿菌、溶血鮑曼不動桿菌、產(chǎn)氣腸桿菌等多種受試菌株均有抑菌活性。對Dybowskin-2CDYa進(jìn)行生物信息學(xué)分析,結(jié)果顯示其為不穩(wěn)定性結(jié)構(gòu)。重組Dybowskin-2CDYa的熱穩(wěn)定性實(shí)驗(yàn)證實(shí),其在100℃加熱5min即可失去抑菌活性。本研究將Dybowskin-2CDYa序列中全部的Arg替換為Lys,并命名為RK6(SAVGKHGKKFGLKKHKKH)。熱穩(wěn)定性試驗(yàn)證明RK6的穩(wěn)定性優(yōu)于Dybowskin-2CDYa�？咕囼�(yàn)證明改造后的RK6,抗菌活性不受影響。利用SOE-PCR方法獲得RK6基因并連接酵母分泌型表達(dá)載體pPICZα-A,構(gòu)建pPICZα-RK6重組表達(dá)質(zhì)粒,利用限制性內(nèi)切酶SacI對重組質(zhì)粒經(jīng)酶切線性化并利用電轉(zhuǎn)化方法轉(zhuǎn)化巴斯德畢赤酵母X33。利用Zeocin篩選陽性轉(zhuǎn)化株,經(jīng)PCR方法驗(yàn)證后進(jìn)行甲醇誘導(dǎo)表達(dá)。對發(fā)酵液上清進(jìn)行抑菌效果測定。結(jié)果顯示:重組抗菌肽RK6對革蘭氏陰性菌(大腸桿菌E.coli)、革蘭氏陽性菌(金黃色葡萄球菌S.au)均有抑制活性。本研究進(jìn)一步構(gòu)建了枯草芽孢桿菌表達(dá)系統(tǒng),將RK6基因的上游連接枯草芽孢桿菌蔗糖果聚糖酶基因(SacB)的啟動子-信號肽序列(SacR)、檢測標(biāo)簽GST-Tag,得到融合序列GSR。將融合序列GSR與枯草芽孢桿菌表達(dá)載體pHY300PLK連接,構(gòu)建表達(dá)質(zhì)粒pHY-GSR。轉(zhuǎn)化枯草芽孢桿菌WB600并利用蔗糖進(jìn)行誘導(dǎo)。結(jié)果顯示:抗菌肽RK6被成功表達(dá)并具備抗菌活性。發(fā)酵液添加飼料中對雛雞進(jìn)行飼喂。動物實(shí)驗(yàn)結(jié)果顯示:飼料中添加發(fā)酵液可提高了雛雞對飼料的利用率、降低雛雞生長的料肉比;雛雞的免疫器官相對重量及血清中部分免疫指標(biāo)(γ-IFN、sIgA、IL-2、LZM)有顯著提高。本文研究結(jié)論:1.從東北林蛙抗菌肽cDNA文庫中篩選得到的11種抗菌肽基因中Dybowskin-2CDYa、Dybowskin-2CDYb 為抗菌肽新家族序列;2.通過抑菌試驗(yàn)證實(shí):Dybowskin-2CDYα對大腸桿菌、金黃色葡糖球菌等多種受試菌均有明顯的抑菌活性;3.將Dybowskin-2CDYα通過氨基酸替換改造成RK6,消除其不穩(wěn)定性,改造后的RK6具有抗菌活性同時熱穩(wěn)定性增加;4.枯草芽孢桿菌表達(dá)系統(tǒng)可以對RK6進(jìn)行表達(dá)。發(fā)酵液應(yīng)用于飼料中可以提高飼喂動物的飼料利用率,并對實(shí)驗(yàn)動物的免疫器官相對重量及血清免疫指標(biāo)水平有明顯的提高。
[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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q78;S816

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