| PA1b is a single chain multifunction peptide, consisted of 37 amino acid residues with 6 cysteines at 3, 7, 15, 20, 22 and 32 of chain positions.It was first isolated from pea seeds In 1986,and it seems to be widespread in legumes. This peptide is thermostable and can intensely resist hydrolysis by trypsin, pepsin and Glu-C proteases in vitro.Up today its functions have been found as follow:1) peros toxicity, it can kill seed weevil, but no harm to human health and the environment;2) It is involved in the regulation of callus growth and cell proliferation;3) it can regulate glucose metabolism When injected at a dose of 5μg/g or more than 5μg/g, PA1b is capable of elevating blood glucose concentration of normal mice and the diabetes type II mice. after subcutaneous injection at a dose of 2.5ug/g, obvious change of blood glucose concentration debase was observed from the diabetes type I ,type II mice.Tertiary structure of PA1b has revealed that PA1b belongs to the cystine-knot peptide family, named cyclotides which were investigated for their potentiality as new antibiotics or anti-HIV properties.This peptide are involved in a growth or metabolic regulation mechanism in a similar fashion to insulin and insulin-like growth factors.it can bind basic 7S globulin(7S Bg) exsisted in soybean seeds,and 7S Bg also can bind insulin and insulin-like growth factors,so it named leginsulin. Leginsulin can catalysis phosphorylation of 7S Bg.To further investigate PA1b distribution and functions ,and also to further clarify the possible link between PA1b and animal insulin systems, monoclonal and polyantibodies against PA1b were prepared as important tool for this peptide researching. PA1b was obtained from pea seeds after purificated and charicaterized And then it was coupled to carrier protein BTG using the two-step glutaraldehyde method as immunal antigen.Using EDC as bridge,it was coupled to carrier protein HSA as screening antigen. Immunized BALB/c mouse with multifocal intradermal injections, spleen cell of immune mice confluence with SP2/0 cell ,screened positive cell of excretion anti-PA1b antibody by ELISA,cloned positive cell by limited dilution method. Five stable hybridoma cell lines producing anti-PA1b MAb were obtained,and named them H9,F6,E5,C7,A10 respectively.by large scale culture , collection ascitic fluid, purification of ascitic fluid,and we obtained purificated monoclonal antibodies. We analyzed the MAb isotypes, titer, specificity, affinity, and found those MAb belong to IgG1 and IgG2b subclass withκlight chain respectively,and have high specificity and affinity.At the same by immunized rabbits,good polyantibodies were obtained.Using these antibodies, we concentrated our efforts on the development of a competitive inhibition ELISA for detection of PA1b Unlike the radioimmunoassay which uses a radiolabeled antigen, the competitive inhibition ELISA uses an enzyme labelled secondary antibody to quantify the antigen. In an competitive inhibition ELISA there is no structural alteration of the antigen.A standard curve was constructed by using serial dilutions of PA1b as standard, which detects a range of 15nmol/L~730nmol/L of PA1b,and approximately 15nmol/L of antigen can be significantly detected.For validation of the ELISA, the intra- and inter-assay variance was determined, by comparison of the reproducibility of the PA1b standard within individual experiments and between experiments, respectively. The mean intra-assay variance of this PA1b ELISA is 2.81% and the mean inter-assay variance is 6.74%, with the recoveries in a range of 97.44～103.84% and 94.87～102.72%, respectivelyBy this kind of ELISA we have investigated distribution of PA1b in many kinds of plant and in many tissues of mice.we found that PA1b not only distributes in many plant,but also in some tissues of mice.furthermore this result is supported by means of immunohistochemical techniques,immunoblott.It is the first to report that the peptide existence across kindom between plant and animal,which expands the new research domain of this peptide.At the same time we found the content of PA1b was changed followed by the sprout of the pea seeds.To investigate the action of PA1b in the regulation of blood sμgar metabolism,with surface plasmon resonance (SPR) biosensor, AFM binding proteins existing in mammalian tissure were studied.and it has been clearly found that a protein in pancreatic cell membrane can bind with PA1b.This binding protein was purified from porcine pancreas by affinity chromatography.The interaction between the purified porcine pancreas proteins and PA1b was further verified by ELISA in vitro.Through SDS-PAGE ,digested in gel by trypsin , MALDI-TOF-MS the peptide mass fingerprint of the protein was obtained. By compared with the data from database (http://www.matrixscience.com) to identify the protein using Mascot search, it was found that this protein is the voltage-dependent anion channel 1 (VDAC 1) and its mass is 30737Da calculated from its amino acid sequence.It is undoubted to bring hope to Clarificate the machenism of PA1b in the regulation of blood sμgar metabolism.
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