百日咳腺苷酸環(huán)化酶毒素基因的克
發(fā)布時(shí)間：2018-09-03 10:49
【摘要】： 目的 通過(guò)對(duì)百日咳桿菌腺苷酸環(huán)化酶毒素基因(cyaA)及cyaC基因進(jìn)行擴(kuò)增,構(gòu)建重組質(zhì)粒pET30a/cyaA、pET30a/cyaC并轉(zhuǎn)化進(jìn)入大腸桿菌DH5α中,經(jīng)篩選保存陽(yáng)性克隆菌。進(jìn)一步構(gòu)建雙基因重組質(zhì)粒pET30a/cyaCA。重組質(zhì)粒pET30a/cyaA轉(zhuǎn)化大腸桿菌BL21(DE3),并使重組百日咳桿菌腺苷酸環(huán)化酶毒素基因cyaA在大腸桿菌BL21(DE3)中表達(dá)。對(duì)表達(dá)的目的蛋白進(jìn)行初步的純化,得到純度達(dá)90%左右的重組蛋白,并對(duì)重組蛋白進(jìn)行初步的鑒定。這些為對(duì)重組百日咳桿菌腺苷酸環(huán)化酶毒素(CyaA, ACT)作進(jìn)一步的應(yīng)用研究奠定基礎(chǔ)。 方法 用試劑盒提取百日咳鮑特菌CS株的總DNA;根據(jù)GenBank收錄的百日咳鮑特菌腺苷酸環(huán)化酶毒素基因cyaA、cyaC基因序列,設(shè)計(jì)引物并送生物公司合成;以所提取的總DNA為模板,PCR擴(kuò)增出cyaA、cyaC基因。對(duì)擴(kuò)增片段和質(zhì)粒進(jìn)行酶切后連接,連接產(chǎn)物轉(zhuǎn)化大腸桿菌DH5α,涂布于卡那平板,37℃培養(yǎng)過(guò)夜,挑取若干菌落少量培養(yǎng),經(jīng)PCR擴(kuò)增鑒定及提取質(zhì)粒后進(jìn)行質(zhì)粒酶切鑒定,陽(yáng)性克隆產(chǎn)物送生物公司測(cè)序。測(cè)序結(jié)果應(yīng)用DNAStar軟件進(jìn)行序列拼接與分析。所得序列在NCBI中進(jìn)行Blast分析。把以pET30a/cyaA為模板PCR擴(kuò)增出的,帶有質(zhì)粒T7啟動(dòng)子的cyaA全片段連接于質(zhì)粒pET30a/cyaC上,同上方法構(gòu)建了重組質(zhì)粒pET30a/cyaCA。 提取陽(yáng)性克隆菌質(zhì)粒pET30a/cyaA,轉(zhuǎn)化大腸桿菌BL21(DE3),涂布于卡那平板,37℃培養(yǎng)過(guò)夜,挑取若干菌落少量培養(yǎng),PCR擴(kuò)增鑒定及提取質(zhì)粒后對(duì)該質(zhì)粒進(jìn)行酶切鑒定。對(duì)陽(yáng)性轉(zhuǎn)化菌進(jìn)行少量培養(yǎng),并用IPTG誘導(dǎo)表達(dá),SDS-凝膠電泳檢測(cè)目的蛋白表達(dá)情況。對(duì)陽(yáng)性轉(zhuǎn)化菌進(jìn)行大量培養(yǎng),IPTG誘導(dǎo)表達(dá)后對(duì)其進(jìn)行純化前處理:超聲破碎菌體、超聲不溶物溶解、SDS-凝膠電泳檢測(cè)目的蛋白表達(dá)形式。用透析液透析待純化液體,上DEAE陰離子交換柱純化蛋白,先用A液(2M urea, 20mM Tris, pH 8.0)洗柱至A280達(dá)基線水平,再用9%B液(2M urea, 2M NaCl, 20mM Tris, pH 8.0)洗脫雜蛋白,最后用40%B液洗脫目的蛋白,收集洗脫峰。SDS-凝膠電泳檢測(cè)洗脫峰含目的蛋白情況,濃縮純化的重組蛋白并分析其純度。 用全細(xì)胞百白破疫苗(wPV)、無(wú)細(xì)胞百白破疫苗(aPV)及生理鹽水免疫NIH小鼠,心臟采血后獲得血清。以純化的重組百日咳CyaA包被96孔板,免疫后小鼠血清作為一抗,采用間接ELISA法檢測(cè)重組百日咳CyaA的反應(yīng)原性。此外,重組百日咳CyaA經(jīng)SDS-凝膠電泳后轉(zhuǎn)移至硝酸纖維素膜,以免疫后小鼠血清作為一抗進(jìn)行Western Blot(WB)檢測(cè),進(jìn)一步檢測(cè)重組百日咳CyaA的反應(yīng)活性。此外,用學(xué)齡前兒童wPV免疫前后血清作一抗對(duì)重組CyaA進(jìn)行ELISA和WB檢測(cè)。 結(jié)果 成功克隆了百日咳腺苷酸環(huán)化酶毒素cyaA基因,其序列已向GenBank提交并被收錄,序列號(hào)為GQ370813。在原核細(xì)胞中表達(dá)了重組百日咳CyaA,經(jīng)初步純化,重組蛋白的純度達(dá)90%左右,濃縮后蛋白濃度為0.83mg/ml。經(jīng)ELISA檢測(cè),全細(xì)胞百白破疫苗免疫組血清的抗體濃度水平明顯高于無(wú)細(xì)胞百白破疫苗和生理鹽水免疫組(p0.05),而無(wú)細(xì)胞百白破疫苗免疫組血清抗體濃度水平與生理鹽水免疫組的無(wú)明顯差異(p0.05)。Western Blot檢測(cè)結(jié)果表明,全細(xì)胞百白破疫苗免疫血清組在預(yù)期的位置有明顯的顯色條帶,無(wú)細(xì)胞百白破疫苗免疫血清組在預(yù)期位置顯色條帶較弱,而生理鹽水免疫血清組則沒(méi)有顯色條帶出現(xiàn)。重組CyaA與學(xué)齡前兒童wPV免疫后血清在ELISA及WB檢測(cè)中均有較強(qiáng)的反應(yīng)。 結(jié)論 在大腸桿菌中成功地克隆了百日咳腺苷酸環(huán)化酶毒素基因cyaA,并構(gòu)建了其原核表達(dá)體系。摸索出重組百日咳腺苷酸環(huán)化酶毒素蛋白的初步純化條件,并對(duì)該重組蛋白進(jìn)行了初步的鑒定,證明其具有良好的反應(yīng)原性。為對(duì)其作進(jìn)一步的應(yīng)用研究奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346
本文編號(hào)：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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346
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