Preliminary Study On Gene Of The Single Chain Antibody Against Swainsonine

The indolizidine alkaloids swainsonine(SW)is the main toxic principal of locoweed,which can result abortion,fetus malformation and male sterility.Locoweed has become an enormous damage on the vast rangeland in China.On the view of nutriology,it is a kind of potential forage grass,it can quickly grow in severe ecological environment. We should regard it as an important part of ecologic environment.The key research is how to control locoweed toxicity and utilize it as excellent grass.TLC,GC and HPLC are effective methods to deter SW.However,SW is insensitive for the common alkaloid developing reagents, which is different from other indolizidine and quinolizidine alkaloids.In addition,GC and HPLC are also for quantitative analysis to deter low content materials.For swainsonine lacks the chromophoric groups,GC and HPLC are not suit for SW analysis.In this study,we attempted to probe the gene of single chain antibody (ScFv) against SW,and hope it could be used to deter and prevent locoism in the future.The results are as follows:1 Comparison of Swainsonine Extraction Technologies from Oxytropis Kansuensis Bunge.The study adopted three extraction technologies industrial ethanol thermal refluxing extraction,hot water extraction and HCl solution extraction,to extract the total alkaloids from Oxytropis Kansuensis Bunge,and silica gel column chromatography to separate the total alkaloids and obtained SW by decompression sublimation.It compared the extraction rates of the alkaloids and SW from Oxytropis Kansuensis Bunge by three technologies.We obtained the white needle crystals,which were identified by TLC,MP,IR,MS and 1HNMR.The comparison indicated that the extraction rates of the SW were separately 18.61μg/g,12.22μg/g,6.23μg/g.The results confirmed that industrial ethanol thermal refluxing extraction was the best technology for SW extraction,which was the most economic and convenient extraction technology.The study triumphantly extracted 500 mg of SW,which provided the raw material foundation for the following study.2 Study on Immunogennicity of SW-HSA on Mice. 12 health Balb/c mice were divided into three groups,group A(4 mice)was immuned with low-dose SW-HSA,group B(4 mice)was immuned with high-dose SW-HSA,group C(4 mice)was control.The number of 3 groups were A1～A4,B1～B4 and C1～C4 respectively,The mice of immune groups were subcutaneously injected in both necks,back,behind legs with CFA emulsied by completely at the first immunization,each mouse in group A was injected 0.05 mg SW-HAS,each mouse in group B was injected 0.10 mg SW-HSA.The second immunization was directed at the 30th day with SW-HSA completely emulsified by FIA,the immune dose and ways were same as the first immunization.The third immunization was done at the 50th day with SW-HSA emulsified by FIA,the immune ways was same as the first time,the dose of group A and group B were 0.075 mg per mouse and 0.15 mg per mouse respectively.The fourth immunization were done at the 70th with SW-HSA dissolved by physiological saline,the immune way were subcutaneously of back and peritoneal injection,the dose of group A and group B were 0.1125 mg per mouse and 0.225 mg per mouse respectively.The results indicated that SW-HSA conjugate could induce immunogennicity in mice.The anti-SW antibody titres were assayed by indirect hemagglutination test and enzyme-linked immunosorbentassay at 10th day after the third immunization and fourth immunization respectively.The results showed that, the titres of first antibody assayed by IHA were 22,21,22,22 in group A and 23,22,22,22 group B,the titres of the second antibody were 27,26,26,26 and 26,25,25,25 in group B,the titres of the first antibody assayed by ELISA were 26,25,25,26 in group A and 27,26,26,26 in group B,the titres of the second antibody were 210,29,210,109 in group A and 29,28,28,28 in group B. After several immunizations,the antibodies titres had increased to 210.The results were the basis for preparing construction the single chain antibody genes against SW.3 Study on Construction Gene of the Single Chain Antibody Against Swainsonine.The study had isolated the total RNA from three mice spleens,and then obtained cDNA of gene groups by reverse transcription system. Referenced to the PCR primers,which were designed by Orlandi.The upstream primers of VH and FR1 of antibody variable region and downstream primers of VL and FR4 of antibody variable region,downstream primers of VH 3' apex and upstream primers of Vκ5' apex were complement sequences.Simultaneously, the primers introduced to restriction enzyme sites in order to connect the carrier and clone antibody variable region gene in prokaryotic and eukaryotic bacteria.By exploring the condition of PCR,The gene fragments of VH and VL were amplified by PCR,and then the VL fragment was amplified by 15 peptide-Linker primers for VL-Linker,the single chain antibody gene was spliced into ScFv gene by SOE-PCR.The results showed that the method improved SOE testing efficiency,The study had amplified the VH,VL,VL-Linker and ScFv gene fragments,the length of fragments were 340 bp,320 bp,420 bp and 760 bp,which were suit for the requirements.The result was the basis for preparing construction the phage antibody library and screening single chain antibody against SW.