Wei sheng yan jiu = Journal of hygiene research · 2006
[Construction and primary identification of a naive Fab fragment phage library].
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Ma Xiao-bing, Chang Ji-wu, Wang Hai-tao, Wang Xin
Аннотация
OBJECTIVE: To construct a naive human Fab fragment phage display library, provide a platform for human antibody preparation and a new therapy for the malignant tumors.
METHODS: Peripheral blood lymphocytes were isolated from 200 ml blood, which was obtained from a healthy blood donor. The heavy chain Fd fragments and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. coli XL1-Blue. The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody of Fabs. The phagemids abstracted from amplified E. coli were cut with endonucleases such as Sac I, Xba I, Spe I and Xho I and amplified by PCR to monitor the insertion of the light chain or heavy chain Fd genes. Human albumin and interleukin-2 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library.
RESULTS: By combination of light chain and heavy chain genes, an antibody library containing 1.2 x 10(6) clones was obtained, and both the cutting of enzymes and PCR showed that there were the light chain or heavy chain Fd genes of the phagemids. After the original antibody library having been panned respectively by two kinds of antigen proteins, it gained enrichment in different degree.
CONCLUSION: Utilizing the technology of phage surface display, special antibody can be gained from the human naive Fab phage display library, which can be used as a new therapy for tumors.
METHODS: Peripheral blood lymphocytes were isolated from 200 ml blood, which was obtained from a healthy blood donor. The heavy chain Fd fragments and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. coli XL1-Blue. The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody of Fabs. The phagemids abstracted from amplified E. coli were cut with endonucleases such as Sac I, Xba I, Spe I and Xho I and amplified by PCR to monitor the insertion of the light chain or heavy chain Fd genes. Human albumin and interleukin-2 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library.
RESULTS: By combination of light chain and heavy chain genes, an antibody library containing 1.2 x 10(6) clones was obtained, and both the cutting of enzymes and PCR showed that there were the light chain or heavy chain Fd genes of the phagemids. After the original antibody library having been panned respectively by two kinds of antigen proteins, it gained enrichment in different degree.
CONCLUSION: Utilizing the technology of phage surface display, special antibody can be gained from the human naive Fab phage display library, which can be used as a new therapy for tumors.