抗白念珠菌天然单链抗体库的构建、筛选及初步鉴定
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摘要
目的:
利用基因工程方法和噬菌体抗体库技术构建人源性白念珠菌单链抗体文库,并从中筛选出特异性的噬菌体单链抗体,为白念珠菌感染的免疫诊断及免疫治疗提供新的方法和手段。
方法:
1 ScFv的连接:取白念珠菌恢复期病人外周血淋巴细胞(PBMC)作为B细胞的来源,提取总RNA,反转录为cDNA,通过降落PCR的方法分别扩增抗体重链可变区(V_H)和抗体轻链可变区(V_L)基因片段。以V_H和V_L基因为模板分别扩增V_H-linker和V_L-linker,然后利用SOE-PCR将他们拼接成完整的单链抗体(ScFv)基因。最后,在单链抗体基因片段的两端引入酶切位点SfiⅠ和NotⅠ。
2 ScFv与噬菌粒载体pCANTAB-5E的拼接及抗体库的构建:单链抗体基因片段与噬菌粒载体pCANTAB-5E各自经SfiⅠ和NotⅠ酶切,连接成重组噬菌体,并转染感受态大肠杆菌XL1-Blue中表达,经辅助噬菌体(helper phage)VCSM13扩增后,构建出初级的噬菌体抗体库。
3噬菌体抗体库的富集筛选:利用白念珠菌标准株与临床分离株对抗体库进行负正筛选,共进行5轮“吸附-洗脱-扩增”的筛选,使抗体库得到有效富集。
4噬菌体ScFv的ELISA鉴定:对第五轮筛选后的阳性克隆的亲和性分别用白念珠菌标准株、临床分离株和大肠杆菌进行ELISA鉴定。
结果:
1从人淋巴细胞中提取的总RNA在1.5%琼脂糖凝胶电泳结果中可见明显的28s、18s和5s条带;V_H基因片段的大小约为340bp,V_L基因片段的大小约为300bp;组装以后的ScFv基因片段约为700bp。
2单链抗体基因片段和pCANTAB-5E分别经SfiⅠ和NotⅠ双酶切后,转化感受态大肠杆菌XL1-Blue,结果获得4.5×10~7cfu/μg氨苄青霉素抗性克隆。随机酶切反应鉴定阳性插入率为83.3%(20/24)。质粒PCR与测序结果证实抗体重链和轻链以整码的单链抗体方式准确的插入载体。
3经过5轮白念珠菌“吸附-洗脱-扩增”的负正性筛选,得到一个4.5×10~7的噬菌体抗体库,说明特异性的ScFv得到了有效富集。
4对第五轮筛选后得到的噬菌体抗体进行ELISA检测,结果显示:3/9的克隆呈阳性反应。
结论:
利用噬菌体抗体库技术,构建了库容量为4.5×10~7的白念珠菌相关人源单链抗体库,并利用白念珠菌临床分离株进行初步筛选。通过ELISA鉴定,获得的噬菌体抗体克隆具有特异性,为白念珠菌感染的临床检测、药物治疗及耐药性的研究提供了理论依据。
Aim:
To construct human phage single-chain antibody library associated with Candida albicans and to screen the specific ScFv in order to supply the new methods for the immunodiagnostics and immunotherapy of candida albieans.
Methods:
1 The ligation of ScFv:
The peripheral blood lymphocytes(PBMC) of candida albicans patients were used as the B cells source,the total RNA of these B cells was extracted and prepared from PBMC of 20 persons,including 10 healthy men,5 newborns and 5 recovering patients infected candida albicans,then reverse transcription for cDNA.First,the V_H and V_L fragments were amplified from the cDNA by touch-down PCR(TD-PCR). Second,the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last,SOE-PCR was used to connect the V_H-linker and V_L-linker,and the SfiⅠand NotⅠrestriction site were inlet in it.PCR products were purified from gel we can get the ScFv.
2 Ligation of ScFv Gene Repertoire into pCANTAB-5E and the phage antibody was constructed:
The gel purified ScFv gene repertoires and pCANTAB-5E were overdigested by SfiⅠand NotⅠseparately,and they were connected to be recombinant phage.The recombinant phage were transfected to competence E.coli XL1-Blue and then to express.By the infection of helper phage VSCM13,the antibody library was constructed.
3 The screening of phage antibody library:
The phage antibody library was panned with human candida albicans and disease candida albicans cell.After five rounds"adsorption-elution-amplified" screening,the specific antibody library was enriched.
4 ELISA was performed to determine the specificity of the phage antibody:
We used human candida albicans,disease candida albicans and E.coli to ELISA. The affinity of positive clones which in the fifth round were determined by ELISA.
Results:
1 The total RNA of these B cells has three bands 28s,18s and 5s in agar gel electrophoresis.V_H fragment is about 340bp,V_L fragment is about 300bp and ScFv is about 700bp.
2 The gel purified ScFv gene repertoires and pCANTAB-5E were overdigested by SfiⅠand NotⅠseparately.The ligation mixes of ScFv and pCANTAB-5E are transfected into competence E.coli XL1-Blue.4.5×10~7cfu/μg ampicillin resistant bacteria colonies growed after overnight culture.Random digestion reaction showed that the positive insert ratio was 83.3%(20/24).Plasmid PCR and sequence showed that variable domain of V_H and V_L were correctly inserted into phagemid vector completely.
3 After 5 rounds of "adsorption-elution-amplified" screening,4.5×10~7 of the phage antibody library to illustrate that the specific ScFv were to be enrichment.
4 The affinity of positive clones which in the fifth round were determined by ELISA,the results showed that:3/9 positive clones have a more than twice fold OD_(405) value control.
Conclusions:
Using phage antibody library display technology,we built a library of 4.5×10~7 for candida albicans-related human single-chain antibody,and we panned with human candida albicans and disease candida albicans.The affinity of positive clones which in the fifth round were determined by ELISA.The specific clones for candida albicans infection in clinical testing,drug treatment and resistance research provides a theoretical basis.
利用基因工程方法和噬菌体抗体库技术构建人源性白念珠菌单链抗体文库,并从中筛选出特异性的噬菌体单链抗体,为白念珠菌感染的免疫诊断及免疫治疗提供新的方法和手段。
方法:
1 ScFv的连接:取白念珠菌恢复期病人外周血淋巴细胞(PBMC)作为B细胞的来源,提取总RNA,反转录为cDNA,通过降落PCR的方法分别扩增抗体重链可变区(V_H)和抗体轻链可变区(V_L)基因片段。以V_H和V_L基因为模板分别扩增V_H-linker和V_L-linker,然后利用SOE-PCR将他们拼接成完整的单链抗体(ScFv)基因。最后,在单链抗体基因片段的两端引入酶切位点SfiⅠ和NotⅠ。
2 ScFv与噬菌粒载体pCANTAB-5E的拼接及抗体库的构建:单链抗体基因片段与噬菌粒载体pCANTAB-5E各自经SfiⅠ和NotⅠ酶切,连接成重组噬菌体,并转染感受态大肠杆菌XL1-Blue中表达,经辅助噬菌体(helper phage)VCSM13扩增后,构建出初级的噬菌体抗体库。
3噬菌体抗体库的富集筛选:利用白念珠菌标准株与临床分离株对抗体库进行负正筛选,共进行5轮“吸附-洗脱-扩增”的筛选,使抗体库得到有效富集。
4噬菌体ScFv的ELISA鉴定:对第五轮筛选后的阳性克隆的亲和性分别用白念珠菌标准株、临床分离株和大肠杆菌进行ELISA鉴定。
结果:
1从人淋巴细胞中提取的总RNA在1.5%琼脂糖凝胶电泳结果中可见明显的28s、18s和5s条带;V_H基因片段的大小约为340bp,V_L基因片段的大小约为300bp;组装以后的ScFv基因片段约为700bp。
2单链抗体基因片段和pCANTAB-5E分别经SfiⅠ和NotⅠ双酶切后,转化感受态大肠杆菌XL1-Blue,结果获得4.5×10~7cfu/μg氨苄青霉素抗性克隆。随机酶切反应鉴定阳性插入率为83.3%(20/24)。质粒PCR与测序结果证实抗体重链和轻链以整码的单链抗体方式准确的插入载体。
3经过5轮白念珠菌“吸附-洗脱-扩增”的负正性筛选,得到一个4.5×10~7的噬菌体抗体库,说明特异性的ScFv得到了有效富集。
4对第五轮筛选后得到的噬菌体抗体进行ELISA检测,结果显示:3/9的克隆呈阳性反应。
结论:
利用噬菌体抗体库技术,构建了库容量为4.5×10~7的白念珠菌相关人源单链抗体库,并利用白念珠菌临床分离株进行初步筛选。通过ELISA鉴定,获得的噬菌体抗体克隆具有特异性,为白念珠菌感染的临床检测、药物治疗及耐药性的研究提供了理论依据。
Aim:
To construct human phage single-chain antibody library associated with Candida albicans and to screen the specific ScFv in order to supply the new methods for the immunodiagnostics and immunotherapy of candida albieans.
Methods:
1 The ligation of ScFv:
The peripheral blood lymphocytes(PBMC) of candida albicans patients were used as the B cells source,the total RNA of these B cells was extracted and prepared from PBMC of 20 persons,including 10 healthy men,5 newborns and 5 recovering patients infected candida albicans,then reverse transcription for cDNA.First,the V_H and V_L fragments were amplified from the cDNA by touch-down PCR(TD-PCR). Second,the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last,SOE-PCR was used to connect the V_H-linker and V_L-linker,and the SfiⅠand NotⅠrestriction site were inlet in it.PCR products were purified from gel we can get the ScFv.
2 Ligation of ScFv Gene Repertoire into pCANTAB-5E and the phage antibody was constructed:
The gel purified ScFv gene repertoires and pCANTAB-5E were overdigested by SfiⅠand NotⅠseparately,and they were connected to be recombinant phage.The recombinant phage were transfected to competence E.coli XL1-Blue and then to express.By the infection of helper phage VSCM13,the antibody library was constructed.
3 The screening of phage antibody library:
The phage antibody library was panned with human candida albicans and disease candida albicans cell.After five rounds"adsorption-elution-amplified" screening,the specific antibody library was enriched.
4 ELISA was performed to determine the specificity of the phage antibody:
We used human candida albicans,disease candida albicans and E.coli to ELISA. The affinity of positive clones which in the fifth round were determined by ELISA.
Results:
1 The total RNA of these B cells has three bands 28s,18s and 5s in agar gel electrophoresis.V_H fragment is about 340bp,V_L fragment is about 300bp and ScFv is about 700bp.
2 The gel purified ScFv gene repertoires and pCANTAB-5E were overdigested by SfiⅠand NotⅠseparately.The ligation mixes of ScFv and pCANTAB-5E are transfected into competence E.coli XL1-Blue.4.5×10~7cfu/μg ampicillin resistant bacteria colonies growed after overnight culture.Random digestion reaction showed that the positive insert ratio was 83.3%(20/24).Plasmid PCR and sequence showed that variable domain of V_H and V_L were correctly inserted into phagemid vector completely.
3 After 5 rounds of "adsorption-elution-amplified" screening,4.5×10~7 of the phage antibody library to illustrate that the specific ScFv were to be enrichment.
4 The affinity of positive clones which in the fifth round were determined by ELISA,the results showed that:3/9 positive clones have a more than twice fold OD_(405) value control.
Conclusions:
Using phage antibody library display technology,we built a library of 4.5×10~7 for candida albicans-related human single-chain antibody,and we panned with human candida albicans and disease candida albicans.The affinity of positive clones which in the fifth round were determined by ELISA.The specific clones for candida albicans infection in clinical testing,drug treatment and resistance research provides a theoretical basis.
引文
[1]赵敬军综述、刘维达 审校:白念珠菌分泌型天冬氨酸蛋白酶(SPA)研究近况,国外医学 微生物学分册,2000,2:27-30
[2]Rolston K Overview of systemic fungal infections.Oncology(Hunting),2001,15Suppl9:11-14
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[4]Bodey GP,Mardani M,Hanna HA,et al.The epidemiology of Candida glabrata and Candida albicans fungemia in immunocompromised patients with cancer.Am J Med,2002,112:380-385
[5]Year H,Sendid B,Francosi N,et al.Contribution of serological tests and blood culture to the early diagnosis of systemic candidiasis.Eur J Clin Microbiol Infect Dis,2001,20:864-870
[6]Laverdiere M,Lalonde RG,Baril JG,et al.Progressive loss of echinocandin activity following prolonged use for treatment of Candida albicans oesophagitis.[J].Antimicrob Chemother,2006,57(4):705-708
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[2]Rolston K Overview of systemic fungal infections.Oncology(Hunting),2001,15Suppl9:11-14
[3]Chen TC,Chen YH,Sai JJ,et al.Epidemiologic analysis and antifungal susceptibility of Candida blood isolates in southern Taiwan.[J].Mocrobiol Immunol Infect,2005,38(3):200-210
[4]Bodey GP,Mardani M,Hanna HA,et al.The epidemiology of Candida glabrata and Candida albicans fungemia in immunocompromised patients with cancer.Am J Med,2002,112:380-385
[5]Year H,Sendid B,Francosi N,et al.Contribution of serological tests and blood culture to the early diagnosis of systemic candidiasis.Eur J Clin Microbiol Infect Dis,2001,20:864-870
[6]Laverdiere M,Lalonde RG,Baril JG,et al.Progressive loss of echinocandin activity following prolonged use for treatment of Candida albicans oesophagitis.[J].Antimicrob Chemother,2006,57(4):705-708
[7]Sobel JD Epidemiology and pathogenesis of recurrent vuvovaginal candidiasis.Am J Obstet Ggnecol.1985,152:925
[8]Morrison SL.Chimeric human antibodymolecules mouse antigen-binding domains with human constant region domains[J].Proc Natl Acad Sci USA,1984,81:6851-6855
[9]Bird RE,Hardaman KD,Jacobson JW,et al.Single chain antigen binding proteins.Science,1988,240(4977):423-426
[10]Yokota Y,Milenic DE,Whitlow.M,et al.Rapid tumor penetration of a single-chain Fv and comparison with other immunoglubin forms[J].Cancer Res,1992,52(12):3402-3408
[11]药立波.医学分子生物学(第2版)[M].北京:人民卫生出版社,2004
[12]Smith GP.Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J].Science,1985,228(4705):1315
[13]Scott JK,Smith JP.Searching for peptide ligands with an epitope library[J].Science,1990,249:386
[14]董志伟,王琰,寿成超.抗体工程(第二版)[M].北京:北京医科大学出版社,2002
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