抗菌肽PR39靶向巨噬细胞表达抗胞内菌感染研究
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摘要
研究背景:
感染性疾病,尤其是慢性持续性感染疾病,是危害动物和人类健康与生命的全球性顽疾之一。其中许多难治且危害严重的感染(如结核、病毒性肝炎等)均与细胞内微生物感染有关。常见的胞内感染细菌主要有:结核杆菌、伤寒沙门氏菌、嗜肺军团菌、麻风杆菌等。这些胞内感染菌进入人体后,能以不同方式逃避巨噬细胞的杀灭并在细胞内生存、繁殖,还能够逃避体液免疫和抗生素的杀灭作用,使巨噬细胞反而成为庇护所,导致临床治疗困难。
抗菌肽(antimicrobial peptides,AMP)是一类具有抗菌活性的多肽,是动物和植物免疫防御系统产生的对抗病原体致病作用的防御性肽类活性物质,在生物体的天然免疫中起着重要的作用。抗菌肽具有广谱、高效的抗菌活性,与其他抗菌药物不易产生交叉耐药性,而且不易诱导耐药菌株的产生。抗菌肽PR39是最初从猪小肠中分离纯化出的一种富含脯氨酸的抗菌肽,是抗菌肽cathelicidin家族中的一员。抗菌肽PR39对细菌(如结核分支杆菌、伤寒沙门氏菌等)具有广谱、高效的抗菌作用。除了抗菌作用以外,PR39还具有中和内毒素、抗炎、免疫诱导、组织修复、抑制凋亡等功能。所以与传统抗生素相比,PR39在细菌感染的治疗中,具有多方面优势,有望成为治疗胞内细菌感染的新一代抗菌药。
然而,抗菌肽在临床实际应用中仍面临着很多问题:①天然抗菌肽分离纯化困难,成本较高,患者不易承受。②作为多肽,口服易被消化破坏,只能静脉或肌注给药,应用不便,并可能引起过敏反应。③容易降解,需要持续给药。④同其他普通抗生素一样,抗菌肽不易在细胞内富集,从而难以发挥其对胞内菌的抗菌作用。因此PR39和其他抗菌肽的临床应用,特别是在胞内菌感染治疗中受到了较大限制。而寻找合理、有效、方便而且经济的治疗方式,成为PR39等新型高效抗菌肽应用于胞内菌感染治疗的关键问题。
研究目的:
构建携带巨噬细胞特异性启动子及抗菌肽PR39基因的重组腺病毒(Ad-SP-PR39),靶向巨噬细胞高效表达抗菌肽PR39基因。从体外细胞水平和体内活体水平检测目的基因在mRNA水平和蛋白水平的表达及基因表达的靶向性,并进一步研究该重组腺病毒在体内外抗胞内菌感染的作用。为抗菌肽PR39在胞内微生物感染基因治疗中的合理安全应用提供理论和实验基础。
研究方法:
1.采用拼接PCR方法合成抗菌肽PR39基因,经NheI和SalI双酶切后克隆入pIRES2-GFP载体,导入小鼠巨噬细胞株RAW264.7,筛选出稳定表达株。通过荧光显微镜、RT-PCR、免疫细胞化学等方法检测抗菌肽PR39基因在小鼠巨噬细胞中的表达情况。采用平板活菌技术方法,检测携抗菌肽PR39基因的小鼠巨噬细胞RAW264.7在体外杀灭胞内菌、抵抗胞内菌感染的能力。
2.抗菌肽PR39多克隆抗体的制备:利用蛋白抗原决定簇预测软件DNASTAR,预测抗菌肽PR39的抗原决定簇序列,用化学合成法合成其抗原决定簇序列。合成的多肽与KLH偶联、纯化后免疫家兔。制备的抗血清经盐析纯化后用SDS-PAGE和ELISA方法进行抗体的纯度和滴度测定。
3.抗菌肽PR39原核表达载体的构建及诱导表达:根据Genbank中抗菌肽PR39序列和大肠杆菌对密码子的偏爱性,设计并合成PR39序列的拼接PCR引物,通过拼接PCR扩增合成PR39 DNA序列。克隆到pET-32a(+)、pET28a、pQE30、pET31b等原核表达载体。在以上载体的基础上,进一步构建了一系列PR39基因的串联表达载体。并对构建的载体进行原核诱导表达。
4.根据GenBank中巨噬细胞特异性启动子序列,设计并合成其拼接引物,通过PCR方法拼接合成巨噬细胞特异性启动子序列。克隆到真核表达载体pEGFP-N1中,替换其中的CMV启动子,得到重组质粒pSP-GFP。将质粒pSP-GFP和pERFP-N1等摩尔浓度混合,用Lipofectamine2000转染小鼠巨噬细胞株RAW264.7、人结肠癌细胞株Lovo、人肝癌细胞株HepG2、人乳腺癌细胞株ZR-75-30和非洲绿猴肾细胞株COS-7。转染48h后用荧光显微镜观察绿色荧光蛋白和红色荧光蛋白的表达并计数。
5.构建由巨噬细胞特异性启动子调控的PR39基因穿梭质粒;将腺病毒骨架质粒pAdEasy-1转入大肠杆菌BJ5183,制备AdEasier Cell并鉴定。将穿梭质粒转化感受态AdEasier Cell,通过细菌内同源重组构建重组腺病毒质粒,进行酶切鉴定。将腺病毒质粒转染293细胞,包装出重组腺病毒Ad-SP-PR39,并扩增和纯化。用重组腺病毒感染小鼠巨噬细胞RAW264.7,在转录水平和蛋白水平检测目的基因的表达。并检测Ad-SP-PR39在各种细胞中表达目的基因的靶向性。
6.用重组腺病毒Ad-SP-PR39体外感染小鼠巨噬细胞RAW264.7,用平板活菌计数方法检测巨噬细胞表达产物对伤寒杆菌和减毒牛型结核分支杆菌(BCG)的杀菌作用。将重组腺病毒经气管感染C57BL/6小鼠肺部,肺组织冰冻切片,荧光显微镜观察腺病毒在肺部的定位及存活情况;采用RT-PCR和免疫荧光技术检测目的基因的表达及定位情况。C57BL/6小鼠经气管感染BCG,感染细菌后再经气管感染重组腺病毒Ad-SP-PR39,并检测腺病毒的表达产物PR39体内对减毒牛型结核分枝杆菌BCG的杀灭效果。
研究结果:
1.用拼接PCR成功扩增出抗菌肽PR39基因,并构建了其真核表达载体pIRES2-PR39,体外转染小鼠巨噬细胞RAW264.7后,筛选出了目的基因的稳定表达株。在细胞中检测到目的基因转录水平和蛋白水平的表达。表达抗菌肽PR39的小鼠巨噬细胞RAW264.7的裂解产物具有较强抗菌作用;携抗菌肽PR39的巨噬细胞体外杀灭胞内伤寒杆菌的能力增强。
2.成功制备了家兔抗PR39多克隆抗体,抗体经盐析纯化后进行SDS-PAGE,电泳结果显示制备的抗体纯度较高。进一步用ELISA方法测定抗体效价,效价达1:10 000。
3.成功构建了抗菌肽PR39基因的多种原核表达载体,反复尝试了目的基因的单独表达、串连表达、与Trx或KSI融合表达、融合后串连表达等多种表达模式,并对诱导表达条件进行多方面优化。但是,由于抗菌肽PR39特殊的抗菌机制和强烈的抗菌活性,导致蛋白产量极低甚至几乎不表达,最终无法得到大量的目的蛋白。
4.采用拼接PCR方法成功合成了巨噬细胞特异性的启动子序列,构建了由巨噬细胞特异性启动子调控的绿色荧光蛋白(GFP)真核表达载体pSP-GFP。质粒pSP-GFP和红色荧光蛋白(RFP)真核表达载体pERFP-N1按1:1混合后,转染巨噬细胞和多种非巨噬细胞。GFP和RFP在不同细胞中的表达具有差异,由巨噬细胞特异性启动子调控的GFP基因仅在巨噬细胞中高效表达,而在非巨噬细胞中则几乎没有表达。而以CMV启动子启动的RFP基因则在巨噬细胞和非巨噬细胞中均有相近的高表达。
5.构建了携巨噬细胞启动子及PR39基因的穿梭质粒和AdEasier Cell。利用细菌内同源重组原理,成功构建成由巨噬细胞特异性启动子调控的PR39腺病毒表达载体(Ad-SP-PR39)。重组腺病毒经扩增纯化后感染小鼠巨噬细胞,能够有效表达目的基因。Ad-SP-PR39能够靶向巨噬细胞有效表达目的基因。
6.重组腺病毒Ad-SP-PR39体外感染小鼠巨噬细胞RAW264.7后,其表达的抗菌肽PR39能增强巨噬细胞对伤寒杆菌和减毒牛型结核分支杆菌(BCG)的杀菌作用。重组腺病毒经气管感染C57BL/6小鼠肺部后,能够有效定位到肺部,并能表达目的基因;采用RT-PCR和免疫荧光技术可以检测目的基因的表达。C57BL/6小鼠经气管感染重组腺病毒Ad-SP-PR39后,PR39基因表达产物能够有效杀灭小鼠肺部的减毒牛型结核分支杆菌BCG。
研究结论:
1.携抗菌肽PR39基因的小鼠巨噬细胞RAW264.7能有效表达抗菌肽PR39,表达产物能增强巨噬细胞体外杀灭胞内伤寒杆菌的能力。
2.成功制备了抗菌肽PR39多克隆抗体。
3.由于抗菌肽PR39特殊的抗菌机制和强烈的抗菌活性,目前尚无法通过原核表达的方式得到大量的目的蛋白。
4.由巨噬细胞特异性启动子调控的GFP基因能够靶向巨噬细胞高效表达。为抗菌肽PR39基因的靶向性表达提供了实验基础。
5.重组腺病毒Ad-SP-PR39能够有效感染小鼠巨噬细胞并表达目的基因,而且目的基因的表达对巨噬细胞具有靶向性。
6.重组腺病毒Ad-SP-PR39能增强巨噬细胞体外杀灭伤寒杆菌和体内外杀灭减毒牛型结核分支杆菌的作用。
Background:
Infectious diseases, especially chronic infection are global diseases threatening animal and human health, of which many refractory and serious infections such as TB, virus hepatitis are involed in intracellular infection. The most common intracellular infectious bacteria include Salmonella Typhi, Mycobecterium Tuberculosis, and Legionella Pneumophila, et al. Infected the body, these bacteria can usually escape the killing of macrophage, live and proliferate in macrophages and escape from the human immunity and antibiotic therapy, so that macrophages become theirs shelter.
Antimicrobial peptide (AMP) as a kind of peptide with antibacterial activity plays an important role in the innate immunity of organisms. It is a kind of defensive peptide with broad-spectrum antibacterial activity, nearly having no cross-resistance with other kinds of antibiotic and can hardly inducing drug-resistance. Antimicrobial peptide PR39 is a proline-rich AMP extracted from pig intestine firstly, which is a member of the cathelicidin family. PR39 has many additional functions such as neutralizing endotoxin, anti-inflammatory effection, immune induction, tissue repairing and inhibiting apoptosis, exepting its broad-spectrum and efficient antibacterial activity to bacteria including Salmonella Typhi and Mycobecterium Tuberculosis. Compared with traditional antibiotics, PR39 has multi-aspect advantage for the therapy of bacterial infection, and it is promising to be a new antibiotic against intracellular bacterial infection.
However, AMPs are facing several handicaps during its clinical application:①It is hard to extract and purify, which cause its high cost not endured by most patients.②As a peptide, AMP would be digested easily in gastrointestinal tract when orally administrated, and could induce allergic reaction when administratded intravenously.③Needing continious administration because of it is easy degradation.④As many other antibiotics, AMP cannot enrich in cells easily, so it is difficult to kill intracellular bacteria. However, it becomes an important problem to find a new efficient, safe and low-costed method to apply AMP in the therapy of intracellular bacteria infection.
Objective:
To construct a recombinant adenovirus vector encoding antimicrobial peptide PR39 gene regulated by a macrophage-specific promoter, and express PR39 effiently and macrophage-specifically in vitro and in vivo. To detect the expression of PR39 gene transcriptionally and translationally in vitro and in vivo, and detect it is macrophage-specifitiy. To furtherly study the protective effects of the recombinant adenovirus encoding PR39 against intracellular bacterial infection in vitro and in vivo. To establish a foundation for the gene therapy of intracellular bacterial infection with antimicrobial peptides.
Methods:
1. RT-PCR method was used to synthesize PR39 gene, which was cloned into pIRES2-GFP vector after digested by NheI and SalI. The recombinant vector was transfected into mouse macrophage RAW264.7 cell, and cells stably expressing PR39 was screened out with G418. The expressions of PR39 in cells were detected with RT-PCR and immunocytochemisty. The capacity of PR39-expressing RAW264.7 cells to kill intracellular bacteria was also investgated.
2. Preparation of PR39 rabbit polyclonal antibody: the epitope including 1-19 amino acids of PR39 was choosened with software DNASTAR and synthesized by chemical method. The synthesized peptide was subjected to immunize rabbit after linked to KLH protein. The antiserum from rabbit was purified with ammonium sulfate salting, and its puritiy and titer were detected with SDS-PAGE and ELISA.
3. Antimicrobial peptide PR39 sequence which was designed according to its sequence in Genbank and the condon preferences of E.coli was synthesized by RT-PCR, and cloned to prokaryotic vector pET-32a (+), pET28a, pQE30, pET31b and so on. The expression of PR39 gene was induced by IPTG.
4. Mrophage-specific promoter sequence, which was designed according to its gene sequence in Genbank, was synthesized by splicing PCR and cloned to eukaryotic vector pEGFP-N1 to substitute its CMV promoter. The recombinant vector pSP-GFP was equimolarly mixed with pERFP-N1 and transfected mouse macrophage cell line RAW264.7, human colorectal cancer cell Lovo, human breast cancer cell line ZR-75-30, human hepatocellular carcinoma cell line HepG2 and african green monkey kidney cell line Cos-7. 48 hours after transfection, the GFP and RFP were observed with fluorescence microscopy.
5. Shuttle vectors encoding PR39 regulated by marophage-specific promoter were constructed and adenovirus backbone plasmids were transformed into E. coli BJ5183 to get AdEasier cell. Recombinant shuttle vectors were then transformed into competent AdEasier cell to construct the recombinant adenovirus plasmid Ad-SP-PR39 through homologous recombination. Recombinant adenovirus plasmids were transfected into packaging cell line 293 to generate adenovirus, which were amplified and then purified further. The recombinant adenovirus was used to infect mouse macrophage RAW264.7 and other non-macrophage cells. The expression of PR39 gene in RAW264.7 was identified by RT-PCR and immunocytochemisty, and the macrophage-specifity was also detected.
6. Recombinant adenovirus Ad-SP-PR39 was used to infect mouse macrophage RAW264.7 in vitro and its antibacterial ability to kill salmonella typhi and Mycobacterium bovis BCG was detected by colony-counting methods. C57BL/6 mice were infected by recombinant adenovirus, fluorescentmicroscopy was used to study the orientation and survival of adenovirus, and RT-PCR and immunofluorescence was used to assay the expression of PR39 gene. C57BL/6 mice were infected by Mycobacterium bovis Bacille Calmette-Gue`rin before they were infected with recombinant adenoviru intratracheally and the antibacterial activity of Ad-SP-PR39 against Mycobacterium was investgated.
Results:
1. PR39 gene was amplified and the recombinant eukaryotic expression vector pIRES2-PR39 was constructed, the vector was transfected into macrophage RAW264.7 to get the PR39-stably-expressing cell line, in which the expression of PR39 gene could be detected both in mRNA and protein levels. Macrophage RAW264.7 expressing PR39 has the capacity to kill intracellular bacteria.
2. Rabbit anti-PR39 polyclonal antiboby was successfully prepared and purified. SDS-PAGE and ELISA showed that it has a high puritiy and titer (1:10000).
3. Some prokaryotic expressing vector encoding PR39 was constructed and the induced expression of PR39 gene was also tried many times, but PR39 gene could hardly be expressed in E.coli. The reasons may be that the expressed PR39 is high toxic to the host, which inhibited its further expression.
4. Macrophage-specific promoter was synthesized by splicing PCR, and pSP-GFP vector regulated by the promoter was then constructed. GFP gene regulated by macrophage-specific promoter was expressed in macrophage cell line RAW264.7, but nearly not in various non-macrophage cell lines, in contrast, the RFP gene regulated by CMV promoter can be expressed in both macrophage cells and non-macrophage cells.
5. Recombiant adenovirus Ad-SP-PR39 encoding PR39 regulated by macrophage-specific promoter was constructed. Mouse macrophage RAW264.7 cell infected by these recombinant adnovirus could express PR39 gene effiently, and recombiant adenovirus Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.
6. Mouse macrophage RAW264.7 infected with recombinant adenovirus Ad-SP-PR39 could express antimicrobial peptide PR39, which enhanced its ability to kill salmonella typhi and Mycobacterium bovis Bacille Calmette-Gue`rin in vitro. Recombinant adenovirus Ad-SP-PR39 could infect C57BL/6 mice efficiently and express antimicrobial peptide PR39. PR39 gene expressed in C57BL/6 mice lung, which were intratracheally infected by recombinant adenovirus Ad-SP-PR39, could kill Mycobacterium bovis Bacille Calmette-Gue`rin given intratracheally.
Conclusions:
1. PR39-gene-transfected mouse macrophage RAW264.7 could express antimicrobial peptide PR39 successfully, which enhances the intracellular antibacterial activity of macrophage cells.
2. Rabbit anti-PR39 polyclonal antibody was successfully prepared.
3. Antimicrobial peptide PR39 could hardly be expressed in prokaryotic host E.coli because of its antibacterial action and toxicity to host cells.
4. GFP gene regulated by macrophage-specific promoter could be expressed in macrophage cells specifically, which suggested the macrophage-specific expression of PR39 gene.
5. Recombinant adenovirus Ad-SP-PR39 could infect mouse macrophage and express PR39 gene efficiently. Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.
6. Recombinant adenovirus Ad-SP-PR39 could enhance the antibacterial ability of macrophage to salmonella typhi in vitro and to Mycobacterium bovis Bacille Calmette-Gue`rin in vitro and in vivo.
感染性疾病,尤其是慢性持续性感染疾病,是危害动物和人类健康与生命的全球性顽疾之一。其中许多难治且危害严重的感染(如结核、病毒性肝炎等)均与细胞内微生物感染有关。常见的胞内感染细菌主要有:结核杆菌、伤寒沙门氏菌、嗜肺军团菌、麻风杆菌等。这些胞内感染菌进入人体后,能以不同方式逃避巨噬细胞的杀灭并在细胞内生存、繁殖,还能够逃避体液免疫和抗生素的杀灭作用,使巨噬细胞反而成为庇护所,导致临床治疗困难。
抗菌肽(antimicrobial peptides,AMP)是一类具有抗菌活性的多肽,是动物和植物免疫防御系统产生的对抗病原体致病作用的防御性肽类活性物质,在生物体的天然免疫中起着重要的作用。抗菌肽具有广谱、高效的抗菌活性,与其他抗菌药物不易产生交叉耐药性,而且不易诱导耐药菌株的产生。抗菌肽PR39是最初从猪小肠中分离纯化出的一种富含脯氨酸的抗菌肽,是抗菌肽cathelicidin家族中的一员。抗菌肽PR39对细菌(如结核分支杆菌、伤寒沙门氏菌等)具有广谱、高效的抗菌作用。除了抗菌作用以外,PR39还具有中和内毒素、抗炎、免疫诱导、组织修复、抑制凋亡等功能。所以与传统抗生素相比,PR39在细菌感染的治疗中,具有多方面优势,有望成为治疗胞内细菌感染的新一代抗菌药。
然而,抗菌肽在临床实际应用中仍面临着很多问题:①天然抗菌肽分离纯化困难,成本较高,患者不易承受。②作为多肽,口服易被消化破坏,只能静脉或肌注给药,应用不便,并可能引起过敏反应。③容易降解,需要持续给药。④同其他普通抗生素一样,抗菌肽不易在细胞内富集,从而难以发挥其对胞内菌的抗菌作用。因此PR39和其他抗菌肽的临床应用,特别是在胞内菌感染治疗中受到了较大限制。而寻找合理、有效、方便而且经济的治疗方式,成为PR39等新型高效抗菌肽应用于胞内菌感染治疗的关键问题。
研究目的:
构建携带巨噬细胞特异性启动子及抗菌肽PR39基因的重组腺病毒(Ad-SP-PR39),靶向巨噬细胞高效表达抗菌肽PR39基因。从体外细胞水平和体内活体水平检测目的基因在mRNA水平和蛋白水平的表达及基因表达的靶向性,并进一步研究该重组腺病毒在体内外抗胞内菌感染的作用。为抗菌肽PR39在胞内微生物感染基因治疗中的合理安全应用提供理论和实验基础。
研究方法:
1.采用拼接PCR方法合成抗菌肽PR39基因,经NheI和SalI双酶切后克隆入pIRES2-GFP载体,导入小鼠巨噬细胞株RAW264.7,筛选出稳定表达株。通过荧光显微镜、RT-PCR、免疫细胞化学等方法检测抗菌肽PR39基因在小鼠巨噬细胞中的表达情况。采用平板活菌技术方法,检测携抗菌肽PR39基因的小鼠巨噬细胞RAW264.7在体外杀灭胞内菌、抵抗胞内菌感染的能力。
2.抗菌肽PR39多克隆抗体的制备:利用蛋白抗原决定簇预测软件DNASTAR,预测抗菌肽PR39的抗原决定簇序列,用化学合成法合成其抗原决定簇序列。合成的多肽与KLH偶联、纯化后免疫家兔。制备的抗血清经盐析纯化后用SDS-PAGE和ELISA方法进行抗体的纯度和滴度测定。
3.抗菌肽PR39原核表达载体的构建及诱导表达:根据Genbank中抗菌肽PR39序列和大肠杆菌对密码子的偏爱性,设计并合成PR39序列的拼接PCR引物,通过拼接PCR扩增合成PR39 DNA序列。克隆到pET-32a(+)、pET28a、pQE30、pET31b等原核表达载体。在以上载体的基础上,进一步构建了一系列PR39基因的串联表达载体。并对构建的载体进行原核诱导表达。
4.根据GenBank中巨噬细胞特异性启动子序列,设计并合成其拼接引物,通过PCR方法拼接合成巨噬细胞特异性启动子序列。克隆到真核表达载体pEGFP-N1中,替换其中的CMV启动子,得到重组质粒pSP-GFP。将质粒pSP-GFP和pERFP-N1等摩尔浓度混合,用Lipofectamine2000转染小鼠巨噬细胞株RAW264.7、人结肠癌细胞株Lovo、人肝癌细胞株HepG2、人乳腺癌细胞株ZR-75-30和非洲绿猴肾细胞株COS-7。转染48h后用荧光显微镜观察绿色荧光蛋白和红色荧光蛋白的表达并计数。
5.构建由巨噬细胞特异性启动子调控的PR39基因穿梭质粒;将腺病毒骨架质粒pAdEasy-1转入大肠杆菌BJ5183,制备AdEasier Cell并鉴定。将穿梭质粒转化感受态AdEasier Cell,通过细菌内同源重组构建重组腺病毒质粒,进行酶切鉴定。将腺病毒质粒转染293细胞,包装出重组腺病毒Ad-SP-PR39,并扩增和纯化。用重组腺病毒感染小鼠巨噬细胞RAW264.7,在转录水平和蛋白水平检测目的基因的表达。并检测Ad-SP-PR39在各种细胞中表达目的基因的靶向性。
6.用重组腺病毒Ad-SP-PR39体外感染小鼠巨噬细胞RAW264.7,用平板活菌计数方法检测巨噬细胞表达产物对伤寒杆菌和减毒牛型结核分支杆菌(BCG)的杀菌作用。将重组腺病毒经气管感染C57BL/6小鼠肺部,肺组织冰冻切片,荧光显微镜观察腺病毒在肺部的定位及存活情况;采用RT-PCR和免疫荧光技术检测目的基因的表达及定位情况。C57BL/6小鼠经气管感染BCG,感染细菌后再经气管感染重组腺病毒Ad-SP-PR39,并检测腺病毒的表达产物PR39体内对减毒牛型结核分枝杆菌BCG的杀灭效果。
研究结果:
1.用拼接PCR成功扩增出抗菌肽PR39基因,并构建了其真核表达载体pIRES2-PR39,体外转染小鼠巨噬细胞RAW264.7后,筛选出了目的基因的稳定表达株。在细胞中检测到目的基因转录水平和蛋白水平的表达。表达抗菌肽PR39的小鼠巨噬细胞RAW264.7的裂解产物具有较强抗菌作用;携抗菌肽PR39的巨噬细胞体外杀灭胞内伤寒杆菌的能力增强。
2.成功制备了家兔抗PR39多克隆抗体,抗体经盐析纯化后进行SDS-PAGE,电泳结果显示制备的抗体纯度较高。进一步用ELISA方法测定抗体效价,效价达1:10 000。
3.成功构建了抗菌肽PR39基因的多种原核表达载体,反复尝试了目的基因的单独表达、串连表达、与Trx或KSI融合表达、融合后串连表达等多种表达模式,并对诱导表达条件进行多方面优化。但是,由于抗菌肽PR39特殊的抗菌机制和强烈的抗菌活性,导致蛋白产量极低甚至几乎不表达,最终无法得到大量的目的蛋白。
4.采用拼接PCR方法成功合成了巨噬细胞特异性的启动子序列,构建了由巨噬细胞特异性启动子调控的绿色荧光蛋白(GFP)真核表达载体pSP-GFP。质粒pSP-GFP和红色荧光蛋白(RFP)真核表达载体pERFP-N1按1:1混合后,转染巨噬细胞和多种非巨噬细胞。GFP和RFP在不同细胞中的表达具有差异,由巨噬细胞特异性启动子调控的GFP基因仅在巨噬细胞中高效表达,而在非巨噬细胞中则几乎没有表达。而以CMV启动子启动的RFP基因则在巨噬细胞和非巨噬细胞中均有相近的高表达。
5.构建了携巨噬细胞启动子及PR39基因的穿梭质粒和AdEasier Cell。利用细菌内同源重组原理,成功构建成由巨噬细胞特异性启动子调控的PR39腺病毒表达载体(Ad-SP-PR39)。重组腺病毒经扩增纯化后感染小鼠巨噬细胞,能够有效表达目的基因。Ad-SP-PR39能够靶向巨噬细胞有效表达目的基因。
6.重组腺病毒Ad-SP-PR39体外感染小鼠巨噬细胞RAW264.7后,其表达的抗菌肽PR39能增强巨噬细胞对伤寒杆菌和减毒牛型结核分支杆菌(BCG)的杀菌作用。重组腺病毒经气管感染C57BL/6小鼠肺部后,能够有效定位到肺部,并能表达目的基因;采用RT-PCR和免疫荧光技术可以检测目的基因的表达。C57BL/6小鼠经气管感染重组腺病毒Ad-SP-PR39后,PR39基因表达产物能够有效杀灭小鼠肺部的减毒牛型结核分支杆菌BCG。
研究结论:
1.携抗菌肽PR39基因的小鼠巨噬细胞RAW264.7能有效表达抗菌肽PR39,表达产物能增强巨噬细胞体外杀灭胞内伤寒杆菌的能力。
2.成功制备了抗菌肽PR39多克隆抗体。
3.由于抗菌肽PR39特殊的抗菌机制和强烈的抗菌活性,目前尚无法通过原核表达的方式得到大量的目的蛋白。
4.由巨噬细胞特异性启动子调控的GFP基因能够靶向巨噬细胞高效表达。为抗菌肽PR39基因的靶向性表达提供了实验基础。
5.重组腺病毒Ad-SP-PR39能够有效感染小鼠巨噬细胞并表达目的基因,而且目的基因的表达对巨噬细胞具有靶向性。
6.重组腺病毒Ad-SP-PR39能增强巨噬细胞体外杀灭伤寒杆菌和体内外杀灭减毒牛型结核分支杆菌的作用。
Background:
Infectious diseases, especially chronic infection are global diseases threatening animal and human health, of which many refractory and serious infections such as TB, virus hepatitis are involed in intracellular infection. The most common intracellular infectious bacteria include Salmonella Typhi, Mycobecterium Tuberculosis, and Legionella Pneumophila, et al. Infected the body, these bacteria can usually escape the killing of macrophage, live and proliferate in macrophages and escape from the human immunity and antibiotic therapy, so that macrophages become theirs shelter.
Antimicrobial peptide (AMP) as a kind of peptide with antibacterial activity plays an important role in the innate immunity of organisms. It is a kind of defensive peptide with broad-spectrum antibacterial activity, nearly having no cross-resistance with other kinds of antibiotic and can hardly inducing drug-resistance. Antimicrobial peptide PR39 is a proline-rich AMP extracted from pig intestine firstly, which is a member of the cathelicidin family. PR39 has many additional functions such as neutralizing endotoxin, anti-inflammatory effection, immune induction, tissue repairing and inhibiting apoptosis, exepting its broad-spectrum and efficient antibacterial activity to bacteria including Salmonella Typhi and Mycobecterium Tuberculosis. Compared with traditional antibiotics, PR39 has multi-aspect advantage for the therapy of bacterial infection, and it is promising to be a new antibiotic against intracellular bacterial infection.
However, AMPs are facing several handicaps during its clinical application:①It is hard to extract and purify, which cause its high cost not endured by most patients.②As a peptide, AMP would be digested easily in gastrointestinal tract when orally administrated, and could induce allergic reaction when administratded intravenously.③Needing continious administration because of it is easy degradation.④As many other antibiotics, AMP cannot enrich in cells easily, so it is difficult to kill intracellular bacteria. However, it becomes an important problem to find a new efficient, safe and low-costed method to apply AMP in the therapy of intracellular bacteria infection.
Objective:
To construct a recombinant adenovirus vector encoding antimicrobial peptide PR39 gene regulated by a macrophage-specific promoter, and express PR39 effiently and macrophage-specifically in vitro and in vivo. To detect the expression of PR39 gene transcriptionally and translationally in vitro and in vivo, and detect it is macrophage-specifitiy. To furtherly study the protective effects of the recombinant adenovirus encoding PR39 against intracellular bacterial infection in vitro and in vivo. To establish a foundation for the gene therapy of intracellular bacterial infection with antimicrobial peptides.
Methods:
1. RT-PCR method was used to synthesize PR39 gene, which was cloned into pIRES2-GFP vector after digested by NheI and SalI. The recombinant vector was transfected into mouse macrophage RAW264.7 cell, and cells stably expressing PR39 was screened out with G418. The expressions of PR39 in cells were detected with RT-PCR and immunocytochemisty. The capacity of PR39-expressing RAW264.7 cells to kill intracellular bacteria was also investgated.
2. Preparation of PR39 rabbit polyclonal antibody: the epitope including 1-19 amino acids of PR39 was choosened with software DNASTAR and synthesized by chemical method. The synthesized peptide was subjected to immunize rabbit after linked to KLH protein. The antiserum from rabbit was purified with ammonium sulfate salting, and its puritiy and titer were detected with SDS-PAGE and ELISA.
3. Antimicrobial peptide PR39 sequence which was designed according to its sequence in Genbank and the condon preferences of E.coli was synthesized by RT-PCR, and cloned to prokaryotic vector pET-32a (+), pET28a, pQE30, pET31b and so on. The expression of PR39 gene was induced by IPTG.
4. Mrophage-specific promoter sequence, which was designed according to its gene sequence in Genbank, was synthesized by splicing PCR and cloned to eukaryotic vector pEGFP-N1 to substitute its CMV promoter. The recombinant vector pSP-GFP was equimolarly mixed with pERFP-N1 and transfected mouse macrophage cell line RAW264.7, human colorectal cancer cell Lovo, human breast cancer cell line ZR-75-30, human hepatocellular carcinoma cell line HepG2 and african green monkey kidney cell line Cos-7. 48 hours after transfection, the GFP and RFP were observed with fluorescence microscopy.
5. Shuttle vectors encoding PR39 regulated by marophage-specific promoter were constructed and adenovirus backbone plasmids were transformed into E. coli BJ5183 to get AdEasier cell. Recombinant shuttle vectors were then transformed into competent AdEasier cell to construct the recombinant adenovirus plasmid Ad-SP-PR39 through homologous recombination. Recombinant adenovirus plasmids were transfected into packaging cell line 293 to generate adenovirus, which were amplified and then purified further. The recombinant adenovirus was used to infect mouse macrophage RAW264.7 and other non-macrophage cells. The expression of PR39 gene in RAW264.7 was identified by RT-PCR and immunocytochemisty, and the macrophage-specifity was also detected.
6. Recombinant adenovirus Ad-SP-PR39 was used to infect mouse macrophage RAW264.7 in vitro and its antibacterial ability to kill salmonella typhi and Mycobacterium bovis BCG was detected by colony-counting methods. C57BL/6 mice were infected by recombinant adenovirus, fluorescentmicroscopy was used to study the orientation and survival of adenovirus, and RT-PCR and immunofluorescence was used to assay the expression of PR39 gene. C57BL/6 mice were infected by Mycobacterium bovis Bacille Calmette-Gue`rin before they were infected with recombinant adenoviru intratracheally and the antibacterial activity of Ad-SP-PR39 against Mycobacterium was investgated.
Results:
1. PR39 gene was amplified and the recombinant eukaryotic expression vector pIRES2-PR39 was constructed, the vector was transfected into macrophage RAW264.7 to get the PR39-stably-expressing cell line, in which the expression of PR39 gene could be detected both in mRNA and protein levels. Macrophage RAW264.7 expressing PR39 has the capacity to kill intracellular bacteria.
2. Rabbit anti-PR39 polyclonal antiboby was successfully prepared and purified. SDS-PAGE and ELISA showed that it has a high puritiy and titer (1:10000).
3. Some prokaryotic expressing vector encoding PR39 was constructed and the induced expression of PR39 gene was also tried many times, but PR39 gene could hardly be expressed in E.coli. The reasons may be that the expressed PR39 is high toxic to the host, which inhibited its further expression.
4. Macrophage-specific promoter was synthesized by splicing PCR, and pSP-GFP vector regulated by the promoter was then constructed. GFP gene regulated by macrophage-specific promoter was expressed in macrophage cell line RAW264.7, but nearly not in various non-macrophage cell lines, in contrast, the RFP gene regulated by CMV promoter can be expressed in both macrophage cells and non-macrophage cells.
5. Recombiant adenovirus Ad-SP-PR39 encoding PR39 regulated by macrophage-specific promoter was constructed. Mouse macrophage RAW264.7 cell infected by these recombinant adnovirus could express PR39 gene effiently, and recombiant adenovirus Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.
6. Mouse macrophage RAW264.7 infected with recombinant adenovirus Ad-SP-PR39 could express antimicrobial peptide PR39, which enhanced its ability to kill salmonella typhi and Mycobacterium bovis Bacille Calmette-Gue`rin in vitro. Recombinant adenovirus Ad-SP-PR39 could infect C57BL/6 mice efficiently and express antimicrobial peptide PR39. PR39 gene expressed in C57BL/6 mice lung, which were intratracheally infected by recombinant adenovirus Ad-SP-PR39, could kill Mycobacterium bovis Bacille Calmette-Gue`rin given intratracheally.
Conclusions:
1. PR39-gene-transfected mouse macrophage RAW264.7 could express antimicrobial peptide PR39 successfully, which enhances the intracellular antibacterial activity of macrophage cells.
2. Rabbit anti-PR39 polyclonal antibody was successfully prepared.
3. Antimicrobial peptide PR39 could hardly be expressed in prokaryotic host E.coli because of its antibacterial action and toxicity to host cells.
4. GFP gene regulated by macrophage-specific promoter could be expressed in macrophage cells specifically, which suggested the macrophage-specific expression of PR39 gene.
5. Recombinant adenovirus Ad-SP-PR39 could infect mouse macrophage and express PR39 gene efficiently. Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.
6. Recombinant adenovirus Ad-SP-PR39 could enhance the antibacterial ability of macrophage to salmonella typhi in vitro and to Mycobacterium bovis Bacille Calmette-Gue`rin in vitro and in vivo.
引文
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[2] Jaklien CL, Theo T, Sebastiaan W, et al. Macrophages play a dual role during pulmonary tuberculosis in mice[J]. J Infect Diseases. 2005.191:65-74
[3] Alamelu Raja. Immunology of tuberculosis[J]. Indian Medical Research. 2004, 120: 213-232
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[7] Vergne I, Chua J, Lee HH, et al. Mechanism of phagelysome biogenesis block viable mycobacterium tuberculosis [J]. Proc Natl Acad Sci USA. 2005,102(11): 4033-4038
[8] Nagabhushanam V, Solache A, Ting LM, et al. Innate inhibition of adaptive immunity: mucobacterium tuberculosis-induced IL-6 inhibits macrophages responses to IFN-gamma[J]. J Immunol. 2003, 171(8):4570-4577
[9] Robert B, Daniel J, Weiner A, et al. Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide. Infect and Immun. 1999, 67(11): 6084-6089
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[11] Julia W, Jun W, Jeanne M, et al. Lack of both type 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by mycobacterium bovis Bacille Calmette-Guerin in IL-12-deficient mice[J]. J Immunol. 1998, 160: 6101-6111.
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[13] Wakeham J, Wang J, Xing Z. Genetically determined disparate innate and adaptive cell-mediated immune responses to pulmonary mycobacterium bovis BCG infection in C57BL/6 and BALB/c mice[J]. Infect. Immun. 2000, 68: 6946-6953
[14] Chen LH, Wang J, Zganiacz A, et al. Single intranasal mucosal mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis[J]. Infect. Immun. 2004, 72(1):238-246
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[1] World Health Organization. The world health reprot: making a difference[R]. 1999:110
[2] Jaklien CL, Theo T, Sebastiaan W, et al. Macrophages play a dual role during pulmonary tuberculosis in mice[J]. J Infect Diseases. 2005.191:65-74
[3] Alamelu Raja. Immunology of tuberculosis[J]. Indian Medical Research. 2004, 120: 213-232
[4] O`Brien L, Roberts B, Andrew PW. In vitro interaction of Mycobacterium tuberculosis and macrophages: activation of anti-mycobacterial activity of macrophages and mechanisms of anti-mycobacterial activity[J]. Curr Top Microbial Immunol. 1996, 215:97-130
[5] Flynn JL, Chan J. Immunology of tuberculosis [J] . Annu Rev Immunol. 2001, 19: 93-129
[6] Kusner DJ. Mechanisms of mycobacterial persistence in tuberculosis[J]. Clin Immunol. 2005, 114(3):239-247
[7] Vergne I, Chua J, Lee HH, et al. Mechanism of phagelysome biogenesis block viable mycobacterium tuberculosis [J]. Proc Natl Acad Sci USA. 2005,102(11): 4033-4038
[8] Nagabhushanam V, Solache A, Ting LM, et al. Innate inhibition of adaptive immunity: mucobacterium tuberculosis-induced IL-6 inhibits macrophages responses to IFN-gamma[J]. J Immunol. 2003, 171(8):4570-4577
[9] Robert B, Daniel J, Weiner A, et al. Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide. Infect and Immun. 1999, 67(11): 6084-6089
[10] Lee PHA, Ohtake T, Zaiou M, et al. Expression of an additional cathelicidin antimicrobial peptide protects against bacterial skin infection[J]. Proc Natl Acad Sci USA. 2005, 102(10): 3750-3755
[11] Julia W, Jun W, Jeanne M, et al. Lack of both type 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by mycobacterium bovis Bacille Calmette-Guerin in IL-12-deficient mice[J]. J Immunol. 1998, 160: 6101-6111.
[12] Cooke MM, Alley MR, Manktelow BW. Experimental infection with BCG as amodel of tuberculosis in the brushtail possum (Trichosurus vulpecula)[J]. New Zealand Veterinary Journal. 2003, 51(3): 132-138
[13] Wakeham J, Wang J, Xing Z. Genetically determined disparate innate and adaptive cell-mediated immune responses to pulmonary mycobacterium bovis BCG infection in C57BL/6 and BALB/c mice[J]. Infect. Immun. 2000, 68: 6946-6953
[14] Chen LH, Wang J, Zganiacz A, et al. Single intranasal mucosal mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis[J]. Infect. Immun. 2004, 72(1):238-246
[1] Boman H G. Antibacterial peptides: Key components needed in immunity[J]. Cell. 1991, 65: 205-207.
[2]石强,刘飞鹏.抗菌肽克隆基因的表达和转基因研究现状[J].生物工程进展. 2000, 20(1): 37-40.
[3] Boman HG, Hultmark D. Insect immunity purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupate of Hyalophora cecropia[J].Eur J biochem. 1980,106:7-16.
[4]赵东红,戴祝英,周开亚.昆虫抗菌肽的功能、作用机理与分子生物学最新进展[J].生物工程进展. 1999, 19(5):14-17.
[5] Steiner H,HuItraark D,Engstrom A,et at.Sequence and specificity of two antibacterial proteins involved in insect immunity[J].Nature. 1981, 292: 246- 248
[6] Boman HG, Beunich S. Sequence and specificity of two antibacteria1.Proteinsinvolved insect immunity[J]. Nature. 1981, 292: 246-248.
[7] Chemysh S, Cocinancich S, Briand J P, et a1. The inducible antimicrobial peptides identification of a unique family of proline-rich peptides and of a novel insect defensin[J]. J Insect Physiology. 1996, 42: 81- 89.
[8] Coinancich S, Dupont A, Hegy G, et a1, Novel inducible antibacterial peptides from a Hemipteran insect, ghesap-sucking bug pyrrholoris apterus[J]. Biochem J. 1994, 300: 567-575.
[9]曾辉明,黄俊生,程立生.昆虫抗菌肽的研究进展[J].热带农业科学. 2002, 22(6): 69-74.
[10]罗刚,魏泓.哺乳动物抗菌肽研究进展[J].四川动物. 2002, 21(4):255-257.
[11] Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor[J]. Proc Natl Acad Sci U S A. 1987, 84(15): 5449–5453
[12]张希春,孙振钧,禚如朋.蚯蚓两种抗菌肽的分离纯化及部分性质[J].生物化学与生物物理进展. 2002, 29(6): 955-959.
[13]康翠洁,王金星,赵小凡,等.中国对虾抗菌肽成熟肽的cDNA克隆[J].山东大学学报. 2002, 37(6): 552-556.
[14]王琼,何清君.植物抗菌肽研究进展[J].四川师范学院学报:自然科学版. 2000, 21(2): 141-145.
[15] Samblanx GW, Codefis IJ, Thevissen K. Mutational analysis of a plant defensin from radish(Raphamus satiwtLs L.) reveals two adjacent for antifuagal activity [J]. J Biol Chem. 1997, 272(2):1171-1179.
[16]胡国平,叶嗣颖.乳链菌肽(Nisin)的研究进展[J].中国微生态学杂志. 2001,13(2):115-117.
[17]顾觉奋,何文杰. Lantibiotic-一类新颖的抗菌肽研究进展[J].国外医药:抗生素册. 2001, 22(6): 274-278.
[18] Oren Z, Shai Y. A class of highly potent antibacterial peptides derived from mosese sole fish pardachirus marmoralus[J]. Eur J Biochem. 1996,237(1):306-
[19]周义文.生物抗菌肽研究进展及应用前景[J].国外医学I临床生物化学与检验学分册. 2004, 25(2): 132-133.
[20] In YP, Chan BP, Mi SK, et a1. Parasin1, an antimicrobial peptide derived fromhistone H2A in the catfish, parasilurus a sotus[J]. FEBS Letters. l998, 437: 258-262.
[21]韦岩.抗菌肽的研究进展和临床应用[J].荷泽医学专科学校学报. 2007, 19(1): 76-78.
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