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Generation and Characterization of Polyclonal Antibodies Against Mouse TIGIt by DNA-Base Immunization

Author: GaoYi
Tutor: ChenZhongHua
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: mTIGIT DNA immunization polyclonal antibodies mouse
CLC: R392
Type: Master's thesis
Year: 2013
Downloads: 6
Quote: 0
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Abstract


Object To generate specificly anti-mouse TIGIT polyclonal antibodies.Methods The mRNA sequence of mouse TIGIT were obtained from GenBank databaseusing bioinformatics method. TIGIT segments were synthesized by RT-PCR from C57mouse spleen. The gene encoding the complete coding sequence (CDS) of mTIGIT wascloned from the total cDNA by PCR. The TIGIT sequence was inserted into the multipleclone sites of pGM-T vector, then subcloned into mammalian expression vector pcDNA3.1(+).After the DNA sequence analysis,the recombinant plasmid TIGIT-pcDNA3.1(+) andthe blank vect pcDNA3.1(+) were amplified and purified. Then the hind legs of rats wereintramuscularly injected with TIGIT-pcDNA3.1(+) and pcDNA3.1(+) vectors at D0、D14and D28,and the blood were harvested at10days after the last immunization. The serumwas purified by Protein A/G PLUS-Agarose affinity chromatography. Western blot andimmunohistochemistry were used to identify the specificity of antibody.Results The rats were successfully immunized. Western blot and immunohistochemistryanalyses revealed that the antibodies generated by DNA immunization can bind with themouse TIGIT. Using these antibodies in immunoblots, TIGIT was detected in lysates of mouse organs. Immunohistochemistry analysis of normal mouse heart、liver、spleen andkidney showed that immunoreactivity was mainly located on endothelial cells of capillaries.Conclusion We successfully generated rat anti-mouse PAbs against mTIGIT by DNAimmunization of rats through intramuscular injection, and provided an effective tool thatcan be used to detect and analyze TIGIT expression and function in mouse systems in thefuture research.

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