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T7 phage capsid protein P11 Expression and purification of monoclonal antibodies

Author: ChenLiang
Tutor: GaoYunYing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: T7 phage P11 protein Prokaryotic expression Monoclonal Antibodies
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 87
Quote: 0
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Abstract


T7 phage infection of E. coli is a potent phage genome is a linear double-stranded DNA, full-length 39 936bp. Including the head of its capsid protein P10A, minor head protein P10B, collar protein P8, tail protein P11, P12 and tail silk protein P17. T7 phage display system as a common , because it has a support capacity, stable insert , washing conditions and flexible , short growth period , etc. are widely used . T7 phage protein chip is the recent development of a novel protein detection methods, high quality, high sensitivity, high specificity and miniaturization characteristics of a protein analysis techniques , enabling foreign protein expressed and demonstrated in bacteriophage T7 surface , and then the display of foreign proteins bacteriophage T7 solidified in the chip, its display to detect proteins . This test is based on P11 gene sequences published in GenBank , we designed a pair of specific oligonucleotide primers by PCR method to obtain the size of 598bp gene fragment and cloned into the expression vector, sequenced successfully constructed pET-28a () / P11 expression vector and transformed into E. coli strain BL21 (DE3) , the induced expression of recombinant protein P11, and further through the Ni-NTA affinity chromatography purified protein purification rate of 90% . Purified product was identified by SDS-PAGE immunized Balb / c mice , with polyethylene glycol PEG -mediated Balb / c mouse spleen cells with SP2 / 0 myeloma cells , with the purified recombinant protein P11 -positive hybridoma screening strains of positive cell clones were cloned by limiting dilution of three screening , ultimately, a stable hybridoma cell lines secreting monoclonal antibody named 2G11. Monoclonal antibodies against subclass identification results showed that 2G11 was IgG2b subclass . ELISA test results and Western blot analysis showed that mAb with bacteriophage T7 protein and P11 protein specific binding . This is for further study of the structure and function of P11 protein , bacteriophage T7 protein chips to improve screening efficiency and the establishment of the foundation phage detection method .

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CLC: > Medicine, health > Basic Medical > Medical Immunology
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