Immunogenicity Diversity Of Germplasm And Its Biological Significance Of ExDNA-ORF's Protein Sc6 In Arthrospira

1.Optimization the method of Western blotting analysis of soluble protein in Arthrospira platensis with Ab-Sc6 as the primary antibodyProteins in Arthrospira platensis had the properties of high content(50%-70%), more kinds and nonuniform abundance,which could badly affect the results of Western blotting analysis.Aiming at this issue,the key experiment conditions of SDS-PAGE and Western blotting had been researched and optimized.Then a kind of Western blotting method with high sensitivity and good reproducibility was established in order to detect the soluble proteins in Arthrospira platensis with Ab-Sc6 as the primary antibody.Sc6 was a kind of protein expressed according to one ORF of extra-genome DNA from Arthrospira platensis Sp-3.The optimized conditions were as follows:(1)In SDS-PAGE analysis,the stacking gel concentration was 5%and electrophoretic voltage was 80 V.And the separation gel concentration was 10%or 12%and electrophoretic voltage was 140 V.(2)Proteins were transferred from gel to NC membrane for 2.5 hours at the conditions of 100 mA constant current.(3)TMB was used as chromogenic substrate.Using the estiblished method,a series of immunogenicity proteins of Sc6 were detected and showed polymorphic diversity within Sp-3 and other four strains of Arthrospira platensis.2.Germplasm immunogenie diversity analysis of Sc6 within Arthrospira Platensis strainsThe results of immunogenicity were analyzed by statistical and clustering method,through detecting soluble proteins from 17 strains of Arthrospira Platensis with the established Western blotting method.The results indicated that the 17 strains were clearly divided into two categories:Sp-1,Sp-2,Sp-6,Sp-9,Sp-10, Sp-13,Sp-14 and Sp-18 formed the first class showing poor production traits in industrialized culture,while Sp-3,Sp-4,Sp-5,Sp-7,Sp-8,Sp-16,Sp-17,Sp-11 and Sp-12 formed the second class showing good production traits.This correlated to our former results using RAPD,16S rRNA,16S-23S ITS,23S rRNA and cpcHID as molecular marker.It was indicated that immunogenicity of Sc6 was closely related to the molecular genetics and production traits of Arthrospira.And it could be conferred that exDNA in Arthrospira had some biological functions,such as environmental adaptation and signal transduction,and was not redundant.The results offered not only a new method to evaluate the production traits of Arthrospira strains,but also a new perspective and approach to deep research and elucidate the origin and evolution of exDNA of Arthrospira and its biological functions.3.Preliminary isolation and purification of immunogenicity protein of Ab-Sc6The content of immunogenicity protein of Sc6 in soluble protein from Arthrospira is very low.Aiming at this issue,hydrogen peroxide was firstly used to decolor some colored substances such as phycobiliprotein.And a native immunogenicity protein in Sp-3 named NS-P1 was detected on the NC membrane. Moreover,by ammonium sulfate precipitation,Sephacryl S-200 gel filtration chromatography and frozen-evaporation concentration,the partly purified NS-P1 was obtained from Sp-3 soluble protein,which had a clear band in the gel stained by coomassie brilliant blue.The results consequently provided the basic for further researching on the purification,MS identification,Structure and functions of NS-P1 and other Sc6 immunogenicity proteins,and revealing the origin and evolution of exDNA in Arthrospira.