百日咳腺苷酸环化酶毒素基因的克
发布时间：2018-09-03 10:49
【摘要】： 目的 通过对百日咳杆菌腺苷酸环化酶毒素基因(cyaA)及cyaC基因进行扩增,构建重组质粒pET30a/cyaA、pET30a/cyaC并转化进入大肠杆菌DH5α中,经筛选保存阳性克隆菌。进一步构建双基因重组质粒pET30a/cyaCA。重组质粒pET30a/cyaA转化大肠杆菌BL21(DE3),并使重组百日咳杆菌腺苷酸环化酶毒素基因cyaA在大肠杆菌BL21(DE3)中表达。对表达的目的蛋白进行初步的纯化,得到纯度达90%左右的重组蛋白,并对重组蛋白进行初步的鉴定。这些为对重组百日咳杆菌腺苷酸环化酶毒素(CyaA, ACT)作进一步的应用研究奠定基础。 方法 用试剂盒提取百日咳鲍特菌CS株的总DNA;根据GenBank收录的百日咳鲍特菌腺苷酸环化酶毒素基因cyaA、cyaC基因序列,设计引物并送生物公司合成;以所提取的总DNA为模板,PCR扩增出cyaA、cyaC基因。对扩增片段和质粒进行酶切后连接,连接产物转化大肠杆菌DH5α,涂布于卡那平板,37℃培养过夜,挑取若干菌落少量培养,经PCR扩增鉴定及提取质粒后进行质粒酶切鉴定,阳性克隆产物送生物公司测序。测序结果应用DNAStar软件进行序列拼接与分析。所得序列在NCBI中进行Blast分析。把以pET30a/cyaA为模板PCR扩增出的,带有质粒T7启动子的cyaA全片段连接于质粒pET30a/cyaC上,同上方法构建了重组质粒pET30a/cyaCA。 提取阳性克隆菌质粒pET30a/cyaA,转化大肠杆菌BL21(DE3),涂布于卡那平板,37℃培养过夜,挑取若干菌落少量培养,PCR扩增鉴定及提取质粒后对该质粒进行酶切鉴定。对阳性转化菌进行少量培养,并用IPTG诱导表达,SDS-凝胶电泳检测目的蛋白表达情况。对阳性转化菌进行大量培养,IPTG诱导表达后对其进行纯化前处理:超声破碎菌体、超声不溶物溶解、SDS-凝胶电泳检测目的蛋白表达形式。用透析液透析待纯化液体,上DEAE阴离子交换柱纯化蛋白,先用A液(2M urea, 20mM Tris, pH 8.0)洗柱至A280达基线水平,再用9%B液(2M urea, 2M NaCl, 20mM Tris, pH 8.0)洗脱杂蛋白,最后用40%B液洗脱目的蛋白,收集洗脱峰。SDS-凝胶电泳检测洗脱峰含目的蛋白情况,浓缩纯化的重组蛋白并分析其纯度。 用全细胞百白破疫苗(wPV)、无细胞百白破疫苗(aPV)及生理盐水免疫NIH小鼠,心脏采血后获得血清。以纯化的重组百日咳CyaA包被96孔板,免疫后小鼠血清作为一抗,采用间接ELISA法检测重组百日咳CyaA的反应原性。此外,重组百日咳CyaA经SDS-凝胶电泳后转移至硝酸纤维素膜,以免疫后小鼠血清作为一抗进行Western Blot(WB)检测,进一步检测重组百日咳CyaA的反应活性。此外,用学龄前儿童wPV免疫前后血清作一抗对重组CyaA进行ELISA和WB检测。 结果 成功克隆了百日咳腺苷酸环化酶毒素cyaA基因,其序列已向GenBank提交并被收录,序列号为GQ370813。在原核细胞中表达了重组百日咳CyaA,经初步纯化,重组蛋白的纯度达90%左右,浓缩后蛋白浓度为0.83mg/ml。经ELISA检测,全细胞百白破疫苗免疫组血清的抗体浓度水平明显高于无细胞百白破疫苗和生理盐水免疫组(p0.05),而无细胞百白破疫苗免疫组血清抗体浓度水平与生理盐水免疫组的无明显差异(p0.05)。Western Blot检测结果表明,全细胞百白破疫苗免疫血清组在预期的位置有明显的显色条带,无细胞百白破疫苗免疫血清组在预期位置显色条带较弱,而生理盐水免疫血清组则没有显色条带出现。重组CyaA与学龄前儿童wPV免疫后血清在ELISA及WB检测中均有较强的反应。 结论 在大肠杆菌中成功地克隆了百日咳腺苷酸环化酶毒素基因cyaA,并构建了其原核表达体系。摸索出重组百日咳腺苷酸环化酶毒素蛋白的初步纯化条件,并对该重组蛋白进行了初步的鉴定,证明其具有良好的反应原性。为对其作进一步的应用研究奠定基础。
[Abstract]:objective
The recombinant plasmid pET30a/cyaA and pET30a/cyaC were constructed by amplifying cyaA and cyaC genes of pertussis bacillus and transformed into E. coli DH5a. The positive clones were screened and preserved. The recombinant plasmid pET30a/cyaCA was further constructed and transformed into E. coli BL21 (DE3) with pET30a/cyaA. The recombinant adenylate cyclase toxin gene cyaA of pertussis bacillus was expressed in E. coli BL21 (DE3). The purity of the expressed protein was about 90% and the recombinant protein was identified preliminarily. Lay the foundation for applied research.
Method
Total DNA of Bolteria pertussis CS strain was extracted by kit, primers were designed and synthesized according to cyaA and cyaC gene sequences of Bolteria pertussis adenylate cyclase toxin collected by GenBank, and cyaA and cyaC genes were amplified by PCR using the extracted total DNA as template. Escherichia coli DH5a was transformed into E. coli DH5a, coated on Kana plate and cultured overnight at 37 C. A few colonies were selected and cultured. The positive clones were identified by PCR amplification and plasmid digestion. The positive clones were sent to biological company for sequencing. The recombinant plasmid pET30a/cyaCA was constructed by the same method.
The plasmid pET30a/cyaA was extracted and transformed into E. coli BL21 (DE3) and coated on Kana plate. Several colonies were cultured overnight at 37 C. The plasmid was identified by PCR amplification and digestion. The transformed bacteria were cultured in small amount and expressed by IPTG. The target protein surface was detected by SDS-gel electrophoresis. The positive transformation bacteria were cultured in large quantities and then purified by IPTG. The bacteria were broken by ultrasonic wave, dissolved by ultrasonic insoluble substance and detected by SDS-gel electrophoresis. At the baseline level of A280, the impurity proteins were eluted with 9% B solution (2M urea, 2M NaCl, 20m Tris, pH 8.0), and the elution peaks were collected with 40% B solution. The elution peaks were detected by SDS-gel electrophoresis, and the purity of the purified recombinant proteins was analyzed.
NIH mice were immunized with whole cell pertussis vaccine (wPV), acellular pertussis vaccine (aPV) and saline to obtain serum after heart collection. The purified recombinant pertussis CyaA was coated with 96-well plate, and the serum of immunized mice was used as an antibody to detect the reactivity of recombinant pertussis CyaA by indirect ELISA. In addition, the recombinant pertussis CyaA was coagulated by SDS-coagulation. Western Blot (WB) assay was used to detect the reactivity of recombinant pertussis CyaA. ELISA and WB were detected by using the sera of pre-school children before and after immunization with wPV.
Result
The cyaA gene of pertussis adenylate cyclase toxin was cloned successfully and its sequence was submitted to GenBank. The recombinant cyaA was expressed in prokaryotic cells. After preliminary purification, the purity of the recombinant protein was about 90% and the concentration of the concentrated protein was 0.83mg/ml. The serum antibody level of group A was significantly higher than that of group B and group B (p0.05), and there was no significant difference between group B and group B (p0.05). The results showed that the staining bands were weaker in the anticipated site in the serum group immunized with ACT vaccine, but not in the serum group immunized with normal saline.
conclusion
CyaA gene of pertussis adenylate cyclase toxin was cloned successfully in E.coli and its prokaryotic expression system was constructed. The preliminary purification conditions of recombinant pertussis adenylate cyclase toxin protein were explored. The recombinant protein was preliminarily identified and proved to have good reactivity. Lay the foundation for applied research.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：2219768
[Abstract]:objective
The recombinant plasmid pET30a/cyaA and pET30a/cyaC were constructed by amplifying cyaA and cyaC genes of pertussis bacillus and transformed into E. coli DH5a. The positive clones were screened and preserved. The recombinant plasmid pET30a/cyaCA was further constructed and transformed into E. coli BL21 (DE3) with pET30a/cyaA. The recombinant adenylate cyclase toxin gene cyaA of pertussis bacillus was expressed in E. coli BL21 (DE3). The purity of the expressed protein was about 90% and the recombinant protein was identified preliminarily. Lay the foundation for applied research.
Method
Total DNA of Bolteria pertussis CS strain was extracted by kit, primers were designed and synthesized according to cyaA and cyaC gene sequences of Bolteria pertussis adenylate cyclase toxin collected by GenBank, and cyaA and cyaC genes were amplified by PCR using the extracted total DNA as template. Escherichia coli DH5a was transformed into E. coli DH5a, coated on Kana plate and cultured overnight at 37 C. A few colonies were selected and cultured. The positive clones were identified by PCR amplification and plasmid digestion. The positive clones were sent to biological company for sequencing. The recombinant plasmid pET30a/cyaCA was constructed by the same method.
The plasmid pET30a/cyaA was extracted and transformed into E. coli BL21 (DE3) and coated on Kana plate. Several colonies were cultured overnight at 37 C. The plasmid was identified by PCR amplification and digestion. The transformed bacteria were cultured in small amount and expressed by IPTG. The target protein surface was detected by SDS-gel electrophoresis. The positive transformation bacteria were cultured in large quantities and then purified by IPTG. The bacteria were broken by ultrasonic wave, dissolved by ultrasonic insoluble substance and detected by SDS-gel electrophoresis. At the baseline level of A280, the impurity proteins were eluted with 9% B solution (2M urea, 2M NaCl, 20m Tris, pH 8.0), and the elution peaks were collected with 40% B solution. The elution peaks were detected by SDS-gel electrophoresis, and the purity of the purified recombinant proteins was analyzed.
NIH mice were immunized with whole cell pertussis vaccine (wPV), acellular pertussis vaccine (aPV) and saline to obtain serum after heart collection. The purified recombinant pertussis CyaA was coated with 96-well plate, and the serum of immunized mice was used as an antibody to detect the reactivity of recombinant pertussis CyaA by indirect ELISA. In addition, the recombinant pertussis CyaA was coagulated by SDS-coagulation. Western Blot (WB) assay was used to detect the reactivity of recombinant pertussis CyaA. ELISA and WB were detected by using the sera of pre-school children before and after immunization with wPV.
Result
The cyaA gene of pertussis adenylate cyclase toxin was cloned successfully and its sequence was submitted to GenBank. The recombinant cyaA was expressed in prokaryotic cells. After preliminary purification, the purity of the recombinant protein was about 90% and the concentration of the concentrated protein was 0.83mg/ml. The serum antibody level of group A was significantly higher than that of group B and group B (p0.05), and there was no significant difference between group B and group B (p0.05). The results showed that the staining bands were weaker in the anticipated site in the serum group immunized with ACT vaccine, but not in the serum group immunized with normal saline.
conclusion
CyaA gene of pertussis adenylate cyclase toxin was cloned successfully in E.coli and its prokaryotic expression system was constructed. The preliminary purification conditions of recombinant pertussis adenylate cyclase toxin protein were explored. The recombinant protein was preliminarily identified and proved to have good reactivity. Lay the foundation for applied research.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
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相关期刊论文 前3条
1 殷大鹏;王华庆;樊春祥;周玉清;梁晓峰;;我国无细胞百白破疫苗纳入免疫规划可行性探讨[J];中国公共卫生管理;2007年03期
2 谢广中;;无细胞百日咳疫苗的发展及应用[J];上海预防医学杂志;2006年01期
3 徐颖华;张庶民;;百日咳的研究现状[J];中华流行病学杂志;2006年08期
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