百日咳腺苷酸環(huán)化酶毒素基因的克
發(fā)布時間：2018-09-03 10:49
【摘要】： 目的 通過對百日咳桿菌腺苷酸環(huán)化酶毒素基因(cyaA)及cyaC基因進行擴增,構建重組質粒pET30a/cyaA、pET30a/cyaC并轉化進入大腸桿菌DH5α中,經(jīng)篩選保存陽性克隆菌。進一步構建雙基因重組質粒pET30a/cyaCA。重組質粒pET30a/cyaA轉化大腸桿菌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。對表達的目的蛋白進行初步的純化,得到純度達90%左右的重組蛋白,并對重組蛋白進行初步的鑒定。這些為對重組百日咳桿菌腺苷酸環(huán)化酶毒素(CyaA, ACT)作進一步的應用研究奠定基礎。 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,設計引物并送生物公司合成;以所提取的總DNA為模板,PCR擴增出cyaA、cyaC基因。對擴增片段和質粒進行酶切后連接,連接產(chǎn)物轉化大腸桿菌DH5α,涂布于卡那平板,37℃培養(yǎng)過夜,挑取若干菌落少量培養(yǎng),經(jīng)PCR擴增鑒定及提取質粒后進行質粒酶切鑒定,陽性克隆產(chǎn)物送生物公司測序。測序結果應用DNAStar軟件進行序列拼接與分析。所得序列在NCBI中進行Blast分析。把以pET30a/cyaA為模板PCR擴增出的,帶有質粒T7啟動子的cyaA全片段連接于質粒pET30a/cyaC上,同上方法構建了重組質粒pET30a/cyaCA。 提取陽性克隆菌質粒pET30a/cyaA,轉化大腸桿菌BL21(DE3),涂布于卡那平板,37℃培養(yǎng)過夜,挑取若干菌落少量培養(yǎng),PCR擴增鑒定及提取質粒后對該質粒進行酶切鑒定。對陽性轉化菌進行少量培養(yǎng),并用IPTG誘導表達,SDS-凝膠電泳檢測目的蛋白表達情況。對陽性轉化菌進行大量培養(yǎng),IPTG誘導表達后對其進行純化前處理:超聲破碎菌體、超聲不溶物溶解、SDS-凝膠電泳檢測目的蛋白表達形式。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。 用全細胞百白破疫苗(wPV)、無細胞百白破疫苗(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法檢測重組百日咳CyaA的反應原性。此外,重組百日咳CyaA經(jīng)SDS-凝膠電泳后轉移至硝酸纖維素膜,以免疫后小鼠血清作為一抗進行Western Blot(WB)檢測,進一步檢測重組百日咳CyaA的反應活性。此外,用學齡前兒童wPV免疫前后血清作一抗對重組CyaA進行ELISA和WB檢測。 結果 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,序列號為GQ370813。在原核細胞中表達了重組百日咳CyaA,經(jīng)初步純化,重組蛋白的純度達90%左右,濃縮后蛋白濃度為0.83mg/ml。經(jīng)ELISA檢測,全細胞百白破疫苗免疫組血清的抗體濃度水平明顯高于無細胞百白破疫苗和生理鹽水免疫組(p0.05),而無細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明顯差異(p0.05)。Western Blot檢測結果表明,全細胞百白破疫苗免疫血清組在預期的位置有明顯的顯色條帶,無細胞百白破疫苗免疫血清組在預期位置顯色條帶較弱,而生理鹽水免疫血清組則沒有顯色條帶出現(xiàn)。重組CyaA與學齡前兒童wPV免疫后血清在ELISA及WB檢測中均有較強的反應。 結論 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,并構建了其原核表達體系。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,并對該重組蛋白進行了初步的鑒定,證明其具有良好的反應原性。為對其作進一步的應用研究奠定基礎。
[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.
【學位授予單位】：廣東藥學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346
本文編號：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.
【學位授予單位】：廣東藥學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346
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