Exploration Of The Methods For Increasing The Effect Of DNA Immunity Of Cecropin-XJ

Cecropin-XJ is a kind of the antimicrobial peptides(AMP) with 37 amino acids and linearα-helix structure. This positively charged small peptide has antibacterial ability of broad spectrum. It can effectively kill Gram-positive and negative bacteria, fungi and tumor cells. The antimicrobial effect on the Gram-positive effect is more stronger than on the Gram-negative bacteria. It would be hard to elucidate uniformly the action mechanism of AMP on the bacteria for its different functions and the ways it works depends on its different structures. To date, there have been five different hypothesis of the action mechanism of AMP on the bacteria and most of which are mainly focused on AMP works on the cell membrane. The functional mechanism of Cecropin-XJ needs to be understood. In order to explore the possible mechanisms, the antibody against the small peptide Cecropin-XJ was raised by nuclear acid coding the Cecropin-XJ prime immunized, nuclear acid coding the Cecropin-XJ boosted. At the same time, Cecropin-XJ was expressed prokaryotically and the recombinant GST-Cecropin-XJ was used to test the titer of antibody against Cecropin-XJ after purification via Glutathione sepharose 4B affinity chromatography. All this work paved the way for study on the action mechanism of Cecropin-XJ on bacteria and testing the transgenic plant with Cecropin-XJ in the future days.The work mainly is composed of the two parts listed below:1. The generation and identification of antibody against Cecropin-XJ by DNA immunization.The gene of interest coding Cecropin-XJ was cut from pGAPza-Cecropin-XJ by restriction enzyme (EcoR I & Not I) with signal peptide nuclear acid sequence. Then, it was subcloned into the eukaryotic expression vector pcDNA3; the recombinant eukaryotic expression vector was named pcDNA3-Cecropin-XJ. Macro prep and purification of pcDNA3-Cecropin-XJ after it was subjected to PCR, restriction enzyme digestion and directly sequenced rightly. It was proved to be expressed with success in mouse's hepatocytes via the hydrodynamics experiment. Then, the Kunming mouse was injected intramuscularly with pcDNA3-Cecropin-XJ 100μg/100μL/per mice at two weeks intervals, the blood was collected every time before immunization. The mouse was subjected to immunize 6th in all. The titer of sera was determined by indirect ELISA. Meanwhile, the purifying Cecropin-XJ was purified via three-step chromatography from Pichia Pastoris yeast. The highest titer sera were chosed to do the immunocolloid gold experiment. The results showed clearly that the Cecropin-XJ works chiefly on the cell membrane of Sa., which is in accordance with the report previously that called'barrel-stave model'.2. The comparision of the method of antibody generationThe gene of interest coding the 4-core sequences of Cecropin-XJ-4R was synthesized chemically with signal peptide sequence was cut from pUC57- Cecropin-XJ-4R using EcoR I & Sal I, then it was subcloned into eukaryotic expression vector pcDNA3, the recombinant eukaryotic expression vector was named pcDNA3-Cecropin-XJ-4R. The eukaryotic expression vector pcDNA3-Cecropin-XJ and pcDNA3-Cecropin-XJ-4R was injected into Kunming mouse intramuscularly respectively, the immune respondence level was compared at last; at the same time, the pcDNA3-Cecropin-XJ-4R was injected into mouse with DNA adjutants pcDNA3-mIL4 and incomplete Freund's adjuvant (IFA), the immune respondence level was compared at last; the mouse was pre-treated with 0.5% hydrochloride procaine and electroporaton (200V/cm), the mouse immune respondence level was compared; the mouse was immuned with pcDNA3-Cecropin- XJ the first three times then boosted with pcDNA3-Cecropin- XJ-4R the second two times and the mouse was immuned with pcDNA3-Cecropin- XJ-4R the first three times then boosted with pcDNA3-Cecropin-XJ the second two times, the mouse immune respondence level was compared at last; meanwhile, the point mutation of gene of interest fragment Cecropin-M8 without signal peptide sequence was subcloned into the prokaryotic expression vector pGEX-4T-1. The GST-Cecropin-M8 was purified with Glutathione sepharose 4B affinity chromatography after expression. The GST-Cecropin-M8 was used to test the antibody against Cecropin-XJ. the effect of immunization of recombinant DNA is best using pcDNA3-Cecropin-4R mixed with pcDNA3-mIL4. In light of high specificity, lower false positiveness, the titer is low, it would be practical and effective way to generate the antibody against Cecropin-XJ using DNA immunization.