The Preliminary Study On Anti-CpTI Antibody Preparation By Nucleic Acid Immunization

The cowpea trypsin inhibitor (CpTt) gene is testified as a broad spectrum insect-resistant gene at present and Its application in insect-resistant botanic transgenic engineering only after S/gene.The CpTI transgenic plant developed rapidly for it's broad spectrum insect- resistant character and the target insects are uneasy tolerance to it. A great deal of modified CpTI transgenic rice was obtained in successfully by our laboratory which represented on a good insect-resistance. Now the biological and molecular assays are the major methods to assay CpTI transgenic plant.There is no better method on assaying the expression of the transgene.The reason would be that the outcome of expression by CpTI is a little peptide and its molecular weight is very small.Its immu-nogenicity would be insufficiency so that It can not induce strong immu-nological reaction and can not produce special antibody of high potency. With the base of related research on nucleic acid immunization, the technology was used to develop the research and application of CpTItransgenic plant. A new antibody preparation method by nucleic acid immunization to assay the expression of the transgenic gene was explored and compared with the traditional one which immune animal by protein.A eukaryotic expression vector pcDNA3.1- CpTl was constructed by insert Cp77 gene into the vector pcDNA3.1 which is used in nucleic acid immunization. The vector was immuned the BALB/c mice by the method intramuscular injection after extracted and purified in great deal. Immu-nological reaction was induced by the expression of CpTl after the vector entered into the mice body. It was testified that the antibody can special immunological recognition the protein GST-CpTI and CpTl by indirect enzyme linked immunosorbent assay(ELISA).The coefficient of correlation is significant and the potency is more than 1:800.For there is no anti-CpTI antibody to use for the assay comparing with the antibody which was obtained by nucleic acid immunization,the work that the CpT/Qene was expressioned in E.co//ar\d purified the protein for antibody preparation was carried out at the same time. Thinking of the CpTl is small and its poor stability, a GST fusion expression system of pGEX was used to improve the immunogenicity of CpTl. Aprokaryotic expression vector pGEX-4T-3- CpTI was constructed by inserted Cp 77 gene into the vector pGEX-4T-3.After expression in E.coli and purified by Glutathione sepharose 4B, the GST-CpTI fusion protein was obtained. By immune the animal with the protein, the antibody which is anti-GST -CpTI fusion protein was prepared.lt was testified that the antibody can special immunological recognition the protein GST-CpTI , GSTand CpTI by indirect ELISA.The coefficient of correlation is significant and The potency is more than 1:8000.The result of assayed the modified GoT/transgenic rice with the two antibodies approved that both of the antibodies can special immunological recognition the protein which roughly extracted from the rice lamina. So ,a new method to preparation anti-CpTI antibody by nucleic acid immunization in assay the expression of C?77transgenic rice can be established.For there are some problems that the potency of the antibody prepared by nucleic acid immunization is not high enough and there is highly background in assay the expression of CpTI transgenic rice,more and further researchs should be carry out.