新型抗菌肽Hadrurin的克隆表达及体内外活性的研究
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摘要
自发现青霉素以来,抗生素一直是人类治疗病原微生物感染疾病的有力武器,但随着抗生素的长期广泛应用,病原菌耐药性的形成日趋严重,因此迫切需要开发新型抗生素,目前从微生物中寻找新生物的难度已愈来愈大。抗菌肽的发现为开发新型抗生素提供了广阔的来源。
抗菌肽(antibacterial peptide)是生物体内经诱导产生的一类具有生物学活性的小分子多肽,广泛存在于多种生物体中,是宿主免疫防御系统的一个重要组成部分,由于其独特的抗菌机制,不会诱发细菌的耐药性,成为具有巨大发展潜力的新型抗菌药物。抗菌肽从天然资源中提取成本高、得率低、工序繁琐;化学合成则价格相当昂贵,也难以应用于临床,利用基因工程技术生产抗菌肽具有重要意义。由于抗菌肽基因一般都较小,故采用人工合成的方法来获取目的基因,再通过基因工程方法进行微生物发酵生产更具有实际意义。本研究的目的就是应用基因工程和蛋白质工程技术研制出重组人源抗菌肽Hadrurin,并研究其体内外抗菌活性,为重组抗菌肽的工程化生产及其在临床上的应用奠定基础。本研究主要从以下几个方面展开。
1选取Genbank中的Hadrurin抗菌肽基因序列,并根据大肠杆菌偏嗜密码子设计并人工合成人源抗菌肽Hadrurin基因,全长136bp。在抗菌肽基因的两端引入肠激酶酶切位点,便于表达后的酶切,保证酶切后具有抗菌肽的天然活性结构。
2 PCR扩增出Hadrurin的基因片段,利用BamHⅠ和SacⅠ限制切位点将基因片段连接到pET32a(+)原核表达载体中。将初步酶切鉴定阳性的质粒测序,验证得到基因序列完全正确的克隆。再将正确的质粒导入大肠杆菌BL21菌株中,用IPTG初步诱导表达,初步确定Hadrurin蛋白可以正常表达且具有较高表达量。
3通过调整诱导时间,诱导剂剂量等方法优化Hadrurin的表达条件。确定诱导4小时,IPTG终浓度0.1mM为Hadrurin的最佳表达条件。并以此条件为基础转为大量诱导表达,将大量诱导表达的Hadrurin蛋白经Ni亲和层析纯化得到纯化的Hadrurin蛋白。
4将纯化的Hadrurin蛋白进行肠激酶酶切,去掉抗菌肽两端的保护性氨基酸恢复其天然活性结构。用MIC和MBC方法计算肠激酶酶切的抗菌肽Hadrurin在不同剂量、不同环境、不同pH值、不同保存温度下对致病性大肠杆菌和金黄色葡萄球菌的抑菌活性及杀菌活性。结果表达Hadrurin对以上两种细菌具有较强的体外抑制活性。
5在体外活性实验的基础上,进行抗菌肽Hadrurin对小鼠的体内保护实验。实验分为预防组和治疗组,结果显示抗菌肽Hadrurin对致病性大肠杆菌的感染具有良好的预防作用,在治疗方面也有一定效果。
Antibiotics,which has been playing a very important role either in the field of animal husbandry or human medicine,is the powerful weapon in the prevention against bacterial infection and the treatment of domestic animal diseases.Besides, antibiotics can also promote the growth of animals as feed additive.However the abuse of antibiotics also brings some serious problems:drug remnant and the emergence of antibiotic-resistant bacterium which is much rapider than the development of new antibiotics.
Antibacterial peptides(ABP) is a kind of peptide that is created by the immunological system of organism to defending the infection of pathogens.It is the important component of innate immunological system.Antibacterial peptides will not induce the resistance of bacteria because of its unique antibacterial machnism,which suggests that ABP can be a potential substitute of traditional antibiotics.
There are three sources for ABP:isolating from natural resources,manual synthesis and expressing through gene engineering techniques.Isolating from natural resources costs heavily and has low productivity with boring procedures.Manual synthesis is very expensive and the preciseness will have negative correlation with the number of amino acid.Gene engineering techniques are prefered to obtain ABP.The purpose of this research is to develop recombinant pig intestine antibacterial pep tide cecropin P1 through the technique of gene engineering and protein engineering and to study the in vitro and in vivo activity,which lay a foundation for the mass production of ABP and the safeguarding of animal husbandry.The study was conducted from following aspects:
1 Using the preferential condon of E.coli,the gene of antibacterial peptide Hadrurin of 136bp was synthesized manually according to the sequence from GenBank. Especially an enterokinase signal cleavage site and glutamine were fused in respectfully 5' and 3' end of the antibacterial peptide genes.
2 The antibacterial peptide gene was cloned into the expression vector pET32a(+) construct the recombineant expression vectors pET-Had.With BamHⅠand SacⅠin the gene of Hadrurin.The clone was sequenccd and induced by using IPTG.
3 Different conditions were detected to adjust the expression of Hadrurin.With temperature and concentration adjusting,4 hours and 0.1mM concentration of IPTG were determined as the best express condition.
4 The antibacterial assay indicated that the peptide dispalyed strong antibacterial activites against Escherichiacoil DH5a,Escherichiacoil O_1,Escherichiacoil O_2 and Staphylococcus aureus.
5 Kunming mouse were used to study the elementary effectiveness of preventing and treating bacterial infection.In the preliminary experiment,the MLD_(100) of Escherichia coil O_1 and Escherichia coil O_2 to mouse were determined.The mice with celiac injection of 240μg purified recombinant antibacterial peptide were detected alive in 48 hours after infection with MLD_(100) of Escherichia coil O_1 and Escherichia coil O_2.
抗菌肽(antibacterial peptide)是生物体内经诱导产生的一类具有生物学活性的小分子多肽,广泛存在于多种生物体中,是宿主免疫防御系统的一个重要组成部分,由于其独特的抗菌机制,不会诱发细菌的耐药性,成为具有巨大发展潜力的新型抗菌药物。抗菌肽从天然资源中提取成本高、得率低、工序繁琐;化学合成则价格相当昂贵,也难以应用于临床,利用基因工程技术生产抗菌肽具有重要意义。由于抗菌肽基因一般都较小,故采用人工合成的方法来获取目的基因,再通过基因工程方法进行微生物发酵生产更具有实际意义。本研究的目的就是应用基因工程和蛋白质工程技术研制出重组人源抗菌肽Hadrurin,并研究其体内外抗菌活性,为重组抗菌肽的工程化生产及其在临床上的应用奠定基础。本研究主要从以下几个方面展开。
1选取Genbank中的Hadrurin抗菌肽基因序列,并根据大肠杆菌偏嗜密码子设计并人工合成人源抗菌肽Hadrurin基因,全长136bp。在抗菌肽基因的两端引入肠激酶酶切位点,便于表达后的酶切,保证酶切后具有抗菌肽的天然活性结构。
2 PCR扩增出Hadrurin的基因片段,利用BamHⅠ和SacⅠ限制切位点将基因片段连接到pET32a(+)原核表达载体中。将初步酶切鉴定阳性的质粒测序,验证得到基因序列完全正确的克隆。再将正确的质粒导入大肠杆菌BL21菌株中,用IPTG初步诱导表达,初步确定Hadrurin蛋白可以正常表达且具有较高表达量。
3通过调整诱导时间,诱导剂剂量等方法优化Hadrurin的表达条件。确定诱导4小时,IPTG终浓度0.1mM为Hadrurin的最佳表达条件。并以此条件为基础转为大量诱导表达,将大量诱导表达的Hadrurin蛋白经Ni亲和层析纯化得到纯化的Hadrurin蛋白。
4将纯化的Hadrurin蛋白进行肠激酶酶切,去掉抗菌肽两端的保护性氨基酸恢复其天然活性结构。用MIC和MBC方法计算肠激酶酶切的抗菌肽Hadrurin在不同剂量、不同环境、不同pH值、不同保存温度下对致病性大肠杆菌和金黄色葡萄球菌的抑菌活性及杀菌活性。结果表达Hadrurin对以上两种细菌具有较强的体外抑制活性。
5在体外活性实验的基础上,进行抗菌肽Hadrurin对小鼠的体内保护实验。实验分为预防组和治疗组,结果显示抗菌肽Hadrurin对致病性大肠杆菌的感染具有良好的预防作用,在治疗方面也有一定效果。
Antibiotics,which has been playing a very important role either in the field of animal husbandry or human medicine,is the powerful weapon in the prevention against bacterial infection and the treatment of domestic animal diseases.Besides, antibiotics can also promote the growth of animals as feed additive.However the abuse of antibiotics also brings some serious problems:drug remnant and the emergence of antibiotic-resistant bacterium which is much rapider than the development of new antibiotics.
Antibacterial peptides(ABP) is a kind of peptide that is created by the immunological system of organism to defending the infection of pathogens.It is the important component of innate immunological system.Antibacterial peptides will not induce the resistance of bacteria because of its unique antibacterial machnism,which suggests that ABP can be a potential substitute of traditional antibiotics.
There are three sources for ABP:isolating from natural resources,manual synthesis and expressing through gene engineering techniques.Isolating from natural resources costs heavily and has low productivity with boring procedures.Manual synthesis is very expensive and the preciseness will have negative correlation with the number of amino acid.Gene engineering techniques are prefered to obtain ABP.The purpose of this research is to develop recombinant pig intestine antibacterial pep tide cecropin P1 through the technique of gene engineering and protein engineering and to study the in vitro and in vivo activity,which lay a foundation for the mass production of ABP and the safeguarding of animal husbandry.The study was conducted from following aspects:
1 Using the preferential condon of E.coli,the gene of antibacterial peptide Hadrurin of 136bp was synthesized manually according to the sequence from GenBank. Especially an enterokinase signal cleavage site and glutamine were fused in respectfully 5' and 3' end of the antibacterial peptide genes.
2 The antibacterial peptide gene was cloned into the expression vector pET32a(+) construct the recombineant expression vectors pET-Had.With BamHⅠand SacⅠin the gene of Hadrurin.The clone was sequenccd and induced by using IPTG.
3 Different conditions were detected to adjust the expression of Hadrurin.With temperature and concentration adjusting,4 hours and 0.1mM concentration of IPTG were determined as the best express condition.
4 The antibacterial assay indicated that the peptide dispalyed strong antibacterial activites against Escherichiacoil DH5a,Escherichiacoil O_1,Escherichiacoil O_2 and Staphylococcus aureus.
5 Kunming mouse were used to study the elementary effectiveness of preventing and treating bacterial infection.In the preliminary experiment,the MLD_(100) of Escherichia coil O_1 and Escherichia coil O_2 to mouse were determined.The mice with celiac injection of 240μg purified recombinant antibacterial peptide were detected alive in 48 hours after infection with MLD_(100) of Escherichia coil O_1 and Escherichia coil O_2.
引文
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