【作者】 王利娜；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本研究旨在构建杂合抗菌肽AT基因的基因工程表达菌,并分别在原核和真核细胞中表达;通过体内、体外抑菌试验、以小鼠为模型的活体试验初步探讨重组抗菌肽AT的生物学功效。首先通过RT-PCR技术,从大肠杆菌攻毒诱导的果蝇中扩增AttacinA基因,通过SOE-PCR技术结合TD-PCR将编码成熟肽的Attacin A基因（简写为mA）与Thanatin基因首位相连连接在一起形成杂合基因Attacin-Thanatin（简写为AT）,构建原核、真核重组表达菌,表达重组杂合抗菌肽AT及mA。利用宿主菌对抗菌肽分子内源攻击的敏感性,即表达的抗菌肽对宿主菌有抑制作用、从而造成宿主菌的部分“自杀”这一原理,建立体内活性检测方法并利用该方法检测杂合抗菌肽AT的体内抑菌活性,同时对原核表达产物初步纯化后进行体外抑菌试验。为了考察重组抗菌肽在活体上的生物学功效,以小鼠为模型初步探讨了真核表达产物在调控生产性能、免疫功能及肠道形态上的效果。主要研究结果如下:1、采用二步法RT-PCR技术,克隆出了Attacin A基因,电泳分析显示,其条带大小约为732 bp,与预期片段大小相符,测序结果表明,其核苷酸序列与GenBank报道的Attacin A基因同源性高达96%～99%以上,证实已经正确克隆获得Attacin A基因。序列分析显示,该基因完整阅读框包含666 bp,编码221 AA。2、以重组TA克隆质粒pMD-AttA为模板,经两次SOE-PCR技术结合TD-PCR扩增后,扩增出杂合肽基因AT,片段大小约为660 bp。该杂合基因以Attacin AN端的189 AA作为N端,C端为Thanatin C端的18 AA,中间有6 AA柔性接头,总共包含213 AA。3、成功构建杂合基因AT及mA的原核重组表达载体pET-mAT（pe）、pET-mA（pe）,转化宿主菌Rosetta-gami^TM（DE3）pLysS,经过IPTG诱导后,SDS-PAGE电泳分析显示,在41.8 kDa、39.2 KDa处有明显的目的条带,与预期目的蛋白分子量理论值一致,表明Rosetta-gami^TM（DE3）pLysS可成功表达AT、mA抗菌肽。4、将重组表达载体pET-mAT（pe）依次转化宿主菌E.coli DH5α、BL21（DE）、Rosetta-gami^TM（DE3）pLysS,采用IPTG诱导表达;通过优化选择该抗菌肽的敏感菌株、诱导剂IPTG浓度、宿主菌诱导起始浓度等初步建立抗菌肽菌内抑菌活性检测方法,并研究了该杂合抗菌肽体内抑菌活性。结果表明以E.coli DH5α为宿主菌、0.1 mmol/LIPTG为工作浓度、D（600nm）值0.3为起始诱导菌浓度检测抗菌肽菌内抑菌活性方法合理可行;AT融合表达产物抑菌活性检测显示,宿主菌E.coli DH5α被本身所表达的杂合抗菌肽所抑制,增殖速度明显较对照菌放缓。5、本试验采用Ni²⁺-NTA agarose beads通过His标签纯化目的蛋白,并初步探讨了该抗菌肽的抑菌活性。结果表明,本试验所获得的抗菌肽AT及mA对E.coli DH5α、E.coli BL21（DE3）、猪霍乱沙门菌、金黄色葡萄球菌具有抑菌活性,其中对大肠杆菌最为敏感。溶血试验表明,其对猪的红细胞几乎没有溶血活性。6、为了检测AT及mA在哺乳动物Vero细胞中的表达特点,试验首先构建增强型绿包荧光蛋白EGFP重组报告载体,通过对细胞绿色荧光的观察来确定该基因在哺乳动物细胞中表达的可行性及表达规律。结果显示,构建的重组真核报告表达载体pEGFP-mAT（ee）、pEGFP-mA（ee）,转染到哺乳动物细胞Vero中后,AT及mA基因均可在Vero细胞中获得绿色荧光蛋白融合表达产物,72 h时融合蛋白表达量最大,绿色荧光定位观察显示,表达的融合蛋白可以分泌到细胞外。试验进一步构建了重组真核表达载体pCI-mAT（ee）、pCI-mA（ee）,转染到Vero细胞中进行表达。根据上述表达特点在转染后72 h检测表达结果,SDS-PAGE电泳显示在23.54 kDa、20.9 kDa处出现目的蛋白条带,大小与预期蛋白分子量理论值基本一致,说明杂合抗菌肽AT及抗菌肽mA均可在Vero细真核胞中获得纯表达。同时收集细胞培养液上清备用。7、通过检测细胞培养液上清对受试菌株的抑菌活性,发现高浓度细胞培养液上清较低浓度的能够更好地抑制细菌的生长,说明该上清中含有目的抗菌肽,可以作为进一步小鼠试验研究的素材。8、选择80只3～4周龄、平均体重为14.2±1.03 g（平均数±标准差）的断奶小鼠随机分为4组,每组5个重复,连续7天腹腔注射含有抗菌肽AT、mA的细胞培养液上清,正常细胞的培养液上清、氨苄西林作为对照。于第8天大肠杆菌攻毒后继续饲养一周。我们发现连续7天腹腔注射含有抗菌肽AT及mA的细胞培养液上清后,AT杂合抗菌肽组小鼠的日增重较其他3组均略有提高,分别比mA组、细胞培养液上清及抗生素组提高了14.7%、13.8%及12.1%,但差异均不显著。而mA组与细胞培养液上清及抗生素组几乎没有差异。攻毒后的一周内,AT杂合抗菌肽组小鼠的生长性能则分别较mA组、细胞培养液上清及抗生素组提高了34.18%、53.62%及17.78%,其中与细胞培养液上清对照组相比差异达到显著水平（p＜0.05）。从试验全期来看,AT杂合抗菌肽组小鼠的日增重与mA组及细胞培养液上清组比较明显提高（p＜0.05）,提高幅度达25%,而与抗生素组差异不显著。连续腹腔注射重组蛋白后,发现其对胸腺、脾脏的相对重量、十二指肠的绒毛高度没有影响,却极显著增加了空肠的绒毛高度,mA组、AT杂合抗菌肽组小鼠分别较细胞培养液上清组升高了77.4%、74.2%（p＜0.01）。对十二指肠隐窝深度有一定的改善,mA组、AT杂合抗菌肽组小鼠分别较细胞培养液上清组下降了16.99%（p＜0.01）、11.12%（p＞0.05）。攻毒后,与对照组相比,重组抗菌肽组小鼠十二指肠、空肠的隐窝深度均较对照组降低,其中AT及mA组小鼠十二指肠隐窝深度分别较抗生素组降低了11.3%（p＜0.05）、15.38%（p＜0.01）;空肠隐窝深度分别较抗生素组降低了4.8%、7.14%,差异均不显著。注射重组抗菌肽后,小鼠血清中的免疫球蛋白IgM、IL-1β的水平明显提高,而攻毒后,IL-1β的水平较对照组下降。结论:本研究成功构建了抗菌肽基因AT、mA的原核、真核基因工程表达菌,并通过体内、体外试验表明了重组抗菌肽AT、mA对大肠杆菌、猪霍乱沙门菌、金黄色葡萄球菌等具有抑菌活性,其中对大肠杆菌最为敏感;通过以小鼠为模型的活体试验证明了该重组抗菌肽在调控小鼠的生产性能、免疫功能及肠道形态上具有较好的生物学功效。更多还原

【Abstract】 There were two aims for this study:（1） to construct the genetic engineering bacteria for expression of the hybrid peptide AT gene;（2） to investigate the biological effectiveness of the recombinant antimicrobial peptide by antibacterial test and in vivo test taking rats as the model.To achieve the above aims,firstly,the full-length open reading frame（ORF） coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template.The hybrid peptide gene Attacin-Thanatin（abbreviated as AT） was generated by using the technology of gene splicing by overlap extension and touchdown PCR.The genetic engineering bacteria for expression of the antimicrobial peptide AT and mA in Escherichia coli and mammalian cell were constructed.According to the principle that host strain is sensitive to antimicrobial peptide,that is to say,the expressed antimicrobial peptide would inhibit the growth of the host strain,thereby the host strain was caused to "suicide",we developed the method to inspect the antimicrobial activity in vivo and the antimicrobial activity of hybrid peptide AT was detected by this method.Meanwhile,the products of prokaryotic expression were purified and antimicrobial test was carried out in vitro.The biological effectiveness of the recombinant peptide AT was studied by in vivo test taking rats as the model,the ability to regulate the growth performance,immune functions and intestinal morphology of weanling rats were inspected.The results are as followings:1.Attacin A gene was cloned by two-step RT-PCR from Drosophila stimulated by E.coli.Results of agarose gel electrophoresis indicated that it included 732 bp in length and encodes 221 AA which was consistent to the predicted size.The sequence analysis revealed that the nucleotide sequence of Attacin A gene had high similarity to other Attacin genes（96%-99%）,which indicated that we have cloned Attacin A gene successfully.The nucleotide sequence of Attacin A gene had been submitted in GenBank（Accession No. EU008826）.2.The hybrid peptide gene AT with the 660bp in length was generated using the technology of splicing by overlap extension and touchdown PCR using the recombinant plasmid pMD-AttA as the template.AT gene included Attacin A（189 amino acids in N terminal） and Thanatin（18 amino acids in C terminal）.In order to avoid affecting their structure,a specific short peptide（-G-G-S-G-S-G-） was added between them.3.The prokaryotic expression vector pET-mAT（pe）,pET-mA（pe） were construted successfully and transformed into Rosetta-gami^TM（DE3）pLysS.E.coli Rosetta cells harbouring the recombinant pET-mAT（pe） and pET-mA plasmid were induced with IPTG and analyzed by SDS-PAGE.Results showed that the molecular mass of recombinant fusion proteins were in agreement with the calculated value from deduced amino acid sequence,which indicated that Rosetta-gami^TM（DE3）pLysS could express the antimicrobial protein AT and mA. 4.To develop the method of inspecting the antimicrobial activity in vivo,the secombinant expression plasmid pET-mAT（pe） was transformed into E.coli DH5α、BL21（DE）、Rosetta-gami^TM（DE3）pLysS respectively.IPTG induction was carried out.In order to optimize the method for assaying bacteriostatic activity in vivo,different factors were tested,including the susceptible strain(E.coli DH5α、BL21（DE3）、Rosetta-gami^TM （DE3） pLysS)、concentration of IPTG（0.0-1.0mM） and the initial bacterial cell density of the host strain（D（600nm） = 0.2-1.0） and so on.Optimal method was obtained at an initial cell density of D（600nm） = 0.3 with 0.1mM IPTG when the most sensitive strain E.coli DH5αwas taken as the host strain.After the recombinant plasmid pET-mAT（pe） was transformed into competent E.coli DH5αand induced with IPTG,cell growth was monitored by measuring the optical density at D（600nm） for continuous 14h.The growth curve demonstrated that the generation of E.coli DH5αharboring recombinant plasmid of pET-mAT（pe） was inhibited obviously compared with the normal E.coli DH5α.5.The His-tag of the fusion protein made it convenient for protein purification by Ni²⁺-NTA agarose affinity chromatography.The antibacterial activity of antimicrobial peptide AT and mA were determined by agarose diffusion assay.The results demonstrated that the recombinant antimicrobial peptide AT and mA were active to E.coli DH5α,E.coli BL21（DE3）,Salmonella choleraesuis and Staphylococcus aureus.Among these test bacteria,E.coli was most susceptible.The toxicities of the peptide to eukaryotic cells were tested by its ability to lyse porcine red blood cells.We found the peptide had almost no detectable hemolytic activity.6.To express AT and mA in mammalian cell,firstly,we carried out the fusion expression with the enhanced green fluorescent protein（EGFP） to determine whether it is feasible to express in mammalian cell through observing the green fluorescent with fluorescent microscope.The results showed that after the recombinant eukaryotic expression vector pEGFP-mAT（ee）、pEGFP-mA（ee） were transfected into Vero cells. There were green fluorescent in Vero cells and the fluorescent amount reached the maximum 72h later.The localization of green fluorescent indicated that the expressed fusion protein could secrete ecto-cell.The eukaryotic expression vector pCI-mAT（ee） and pCI-mA（ee） were constructed subsequently and transfected into Vero cells. According to the above results,SDS-PAGE was conducted after 72h of transfection. Results showed that there were predominant bands with apparent molecular weight of 23.54 kDa and 20.9 kDa which was consistent with the molecular weight calculated from the amino acid sequence.So,eukaryotic expression in Vero cells were carried out successfully.The supernatant of cell culture fluid were collected for the following usage.7.By testing the antibacterial activity to E.coli DH5α,E.coli JM109,Streptococcus, Staphylococcus aureus and Salmonella choleraesuis of the supernatant of cell culture medium containing the recombinant peptide AT or mA,we found that the higher concentration of the supernatant,the more inhibition of the bacteria,which indicated that the supernatant of cell culture medium contained the antimicrobial peptide AT or mA and could be as the material for the further study on rats.8.Eighty weanling rats with an average initial weight of 14.2±1.03g（means±SD） were randomly allocated to four treatments with five replicate per treatment.The supernatant of cell culture medium containing the recombinant peptide AT or mA was administered by intraperitoneal injection into rats for 7 days consecutively.At the same time,the supernatant of cell culture medium of normal cell and Ampicillin were as control.On day 8, the fresh E.coli was administered by intraperitoneal injection into rats and the rats were reared for another week.The results showed that the supernatant of cell culture medium containing the recombinant peptide AT slightly increased the average daily gain（ADG） of rats by 14.7%、13.8%and 12.1%compared with the supernatant of cell culture medium containing the recombinant peptide mA,the supernatant of cell culture medium of normal cell and Ampicillin.But the difference was not significant.There was no different effect on ADG among the supernatants of cell culture medium containing the mA and of normal cell and Ampicillin.After infected with E.coli,the growth performance of rats treated with the supernatant containing the recombinant peptide AT increased 34.18%、53.62%（p＜0.05） and 17.78%compared with the supernatant containing the recombinant peptide mA and of normal cell and Ampicillin.From the whole experiment period,the ADG of rats treated with the supernatant containing the recombinant peptide AT increased significantly compared with the supernatant containing the mA and of normal cell（p＜0.05）,but there was no significant difference compared with Ampicillin.The supernatant containing the recombinant peptide AT or mA did not affect the relative weights of immune organ thymus gland and spleen,villus height of duodenum,but the villus height of jejunum increased by 74.2%（p＜0.01） and 77.4%compared with the supernatant of normal cell.Meanwhile,crypt depths of duodenum were decreased by 11.12%（p＞0.05） and 16.99%（p＜0.01） compared with the supernatant of normal cell.After infected with E.coli,the crypt depth of duodenum of rats treated with the supernatant containing the recombinant peptide AT or mA were decreased by 11.3%（p＜0.05）,15.38%（p＜0.01） while the crypt depth of jejunum decreased by 4.8%,7.14%compared with Ampicillin.In addition,the level of IgM、IL-1βin serum of rats treated with the supernatant containing the recombinant peptide AT or mA increased obviously while the level of IL-1βdecreased after infected with E.coli.Conclusions:The genetic engineering bacteria for expression of the antimicrobial peptide AT and mA in Escherichia coli and mammalian cell were constructed successfully. Antibacterial test indicated that antimicrobial peptide AT and mA were active to E.coli, Salmonella choleraesuis,Staphylococcus aureus and so on.Among these test bacteria, E.coli was most susceptible.In vivo experiments taking the rats as the model demonstrated that the recombinant antimicrobial peptide AT possessed the biological effectiveness on regulating the growth performance,immune functions and intestinal morphology of weanling rats.更多还原