| BackgroundAs is well known,the boundaries of space is so vast that the ground can not match the environmental conditions.It was studied that the environments of space have the features of strong radiation,high vacuum,micro-gravity,high cleanliness,etc and there exists the factors that can lead to biology-induced mutations.Early in the 60's of 20th century,the United States,the Soviet Union and other scientists had found the influence of the space environment on the viruses,bacteria,plant chromosome and they found that radiation might cause the characteristics of DNA strand breaksabnormal cell,differentiation,high space radiation-induced mutation rate in a variety of organisms.Then the space powers of the world have explored the influence of the space mutagenic effects on the other life substances.The studies were about mutagenic compounds traits,functional studies at the macro level and protein and gene researches at the micro level.They confirmed that the space environment can not only induce to modify traits of organisms and the functions but also can induce the gene mutations and affect protein expressions.Complex sugars play important parts in biology division,reproduction,signal transduction and so on.but So far,the researches that the influence of expression of polysaccharide molecule in space environment is still less.As a new field,the space environment has become the world's hot spots in space science researches and it may play an important role in energy,pharmaceutical and many other fields.Relevant data shows that the pharmaceutical space equivalent to one month's production can be 30-60 years on earth production.there are several drugs that after the satellite delivering has been very good results in our country.This shows that the use of space mutagenic effects will be a very broad prospect for Pharmaceuticals.Polysaccharide molecules are important parts of organisms including animal,plant material,microbes and all life subst.it has the functions of maintaining essential life,composition of skeletal frame, portfolio receptors,immune regulation,and many other biological activities. Microorganisms such as bacteria that exist many polysaccharide molecules,and it has very important functions.According to many studies,it has a wide range of biological activities in stimulating the bone marrow,causing macrophage proliferation and apoptosis,the production of polysaccharide vaccine,antibody and treatment of various diseases.α-hemolytic streptococcus as a Streptococcus species,also known as alpha-hemolytic streptococcus,the opportunistic pathogen,its fermentation product glycopeptides has high biological value including anti-tumor,stimulating the bone marrow hematopoietic and areas such as regulating autoimmune.Lectins are a class of nonimmunogenicity,to identify sugar chain stru-cture of proteins.Its specificity of binding is similar to antigen-antibody reaction.Lectins can be divided into seven categories:(1) D-mannose,or(D-glucose) group;(2) N-acetyl glucosamine group;(3) N-acetyl galactose group;(4) D-galactose;(5) L-fucose group;(6) sialic acid group;(7) group of complex sugars.Lectins have many important biological activities and it plays an important role in anti-nutritional, defensive functions,cell maturation,differentiation markers,tumor diagnosis and immunohistochemistry.In addition,the plant lectin as an important natural products,is an important tool to separate and purificate glycoconjugates and study the structure and function of glycoconjugates.It is also an ideal model to study the mechanism of interaction between sugar and protein.And it will have a wide range of applications in transgenic insect-resistant,biological nitrogen fixation,drug development,bone marrow transplantation,disease identification and so on.ObjectiveWe select a representative lectin at different types of lectin-class,namely,ConA, WGA,SNA,RCA-Ⅰand PHA.And they are marked by the horseradish peroxidase through improved heptaiodic acid sodium mathods.Using of lectin-binding characteristics,we detect the levels of polysaccharide molecular in Streptococcus through five kinds of lectins.In order to know the levels of expression of polysaccharide molecular in Streptococcus before and after the effection of space environment to explore whether the space environment can regulate the expression of polysaccharide molecular.Whether it can provide a new way for polysaccharide biological products screening and enhancing performance or improving production and whether we can provide a new experiental evidence of the biological functions for the space mutation bacteria.Materials And Methods1.Bacteria Space Streptococcus species are provided by Shanxi Space Biopharmaceutical Engineering Research Center.We select the species with high yield of mutagen sensitivity and wide variation of strain rate,and they were Placed in medium package back into the cabin of Shenzhou spacecraft to carry out a few days in orbit(perigee altitude of 200 km,apogee height 350 km).And after several mutagenesis of the space environment,we repeatedly selected strains that were variations in space.The above two strains were inactivated after cobalt 60 with irradiation 9000GY,stored in -80℃refrigerator.2.The broken strains,extraction of proteoglycan molecules We use ultrasonic fragmentation method to broken bacteria,the centrifugal supematant check,then use salt-soluble method to extract protein polysaccharide molecules,the supernatant by adding ammonium sulfate to saturation at 60%～90%,Continue mixing 10～30 min,4℃standing 3～5 h,centrifuged and the precipitate dissolved in distilled water. using Sevage method to remove free protein,the centrifugal supernatant collected, Rotary Evaporator Removal of residual solvent,4℃dialysis,freeze-drying,and a protein polysaccharide powder are acquired.3.Lectin marking Lectins are marked by the horseradish peroxidase through improved heptaiodic acid sodium mathods and then they are filtered by Sephadex G200. We use protein SDS-PAGE gel electrophoresis to detect the effect after affinity chromatography and then measure the rate of enzyme markers and ELISA antibody titers.4.Polysaccharide Molecules Detected detection methods Proteoglycan molecule-HRP labeled lectin method(similar to direct ELISA),we use 96 coated ELISA plates to coat protein polysaccharide molecules,add tags agglutinin HRP-best, fully integrated,not-eluting combination agglutinin HRP,TMB-H2O2 solution through color,measuring absorbance.If bacterial polysaccharide molecules is more and the combination of HRP labeled lectins is more.Then the OD is high after colored and the OD is lower on the contrary.5.All OD values of the data are expressed by mean±standard deviation((?)±s) express.We used statistical package for social science(SPSS)13.0 for analysis,and used two-sample t-test.All results were statistically significant for 0.05.if P＜0.05 that the difference was statistically significant,if P＞0.05 that the difference was not statistically significant. Results1.The broken strains,extraction of proteoglycan moleculesAccording to the formula:relative bacterial broken rate =(1 - precipitation of bacterial wet weight after ultrasound / precipitation of bacterial wet weight before ultrasonography),yield of proteoglycan = proteoglycan / bacterial weight×100%; the bacteria relative fragmentation rate was 99%.Yield of Proteoglycan:ground Streptococcus space Streptococcus2.Lectins markingHRP-lectin-labeled rates were:OD403nm/OD280nm 0.73,0.78,0.75,0.73 and 0.75;ELISA titer measured results:1:1000,1:000,1:1000,1:800 and 1:600.3.Polysaccharide Molecules Detected(1) The OD of expression of the ConA Related Polysaccharide with High Mannose in common ground streptococcus is 0.97±0.05;The OD of expression of the ConA Related Polysaccharide with High Mannose in space streptococcus is 1.18±0.03;the OD value of Positive control group is 1.46±0.05,the OD value of negative control group is 0.05±0.00,Compare of the two strains,P values is 0.000 and it is less than 0.05,they have statistical significance.(2) The OD value of expression of the WGA Related with Terminal GlcNAc in common ground streptococcus is 1.16±0.04;The OD value of expression of the WGA Related with Terminal GIcNAc in space streptococcus is 1.16±0.03;the OD value of Positive control group is 1.46±0.03,the OD value of negative control group is 0.05±0.00,Compare of the two strains,P values is 0.967 and they have no statistical significance.(3) The OD value of expression of the SNA Related Polysaccharide with Terminal Neu5Aca2-6Gal in common ground streptococcus is 0.56±0.03;The OD value of expression of the SNA Related Polysaccharide with Terminal Neu5Aca2-6Gal in space streptococcus is 0.75±0.03;the OD value of Positive control group is 1.35±0.04,the OD value of negative control group is 0.05±0.00,Compare of the two strains, P values is 0.000 and it is less than 0.05,they have statistical significance.(4) The OD value of expression of the RCA-ⅠRelated Polysaccharide with Terminal Galβ1-4 GlcNAc in common ground streptococcus is 1.19±0.06;The OD value of expression of the RCA-ⅠRelated Polysaccharide with Terminal Galβ1-4 GlcNAc in space streptococcus is 1.38±0.08;the OD value of Positive control group is 1.42±0.05,the OD value of negative control group is 0.05±0.00,Compare of the two strains,P values is 0.001 and it is less than 0.05,they have statistical significance.(5) The OD value of expression of the PHA Related Polysaccharide with Terminal Gal-NAC in common ground streptococcus is 0.85±0.06;The OD value of expression of the PHA Related Polysaccharide with Terminal Gal-NAC in space streptococcus is 0.84±0.05;the OD value of Positive control group is 1.44±0.06,the OD value of negative control group is 0.05±0.00,Compare of the two str-ains,P values is 0.664 and they have no statistical significance.ConclusionsIn this experiment,we use the improved heptaiodic acid sodium mathods to mark all the lectins by horseradish peroxidase,use ultrasonic fragmentation method to broken bacteria,use salt-soluble method to extract protein polysaccharide molecules.And then we use the principle that the lectins can detect polysaccharide molecules specially.Lectins are a class of non-immunogenicity,to identify sugar chain structure of proteins.Its specificity of binding is similar to antigen-antibody reaction.Using the lectins marked by horseradish peroxidase to detect the levels of polysaccharide molecular in Streptococcus through five kinds of lectin.We use the characteristics of the ConA Related Polysaccharide with High Mannose,the WGA Related with Terminal GlcNAc,the SNA Related Polysaccharide with Terminal Neu5Aca2-6Gal,the RCA-ⅠRelated Polysaccharide with Terminal Galβ1-4 GlcNAc,the PHA Related Polysaccharide with Terminal Gal-NAC to detect the differences of the expression of polysaccharide molecular between the general groundα-hemolytic and the space streptococcus.The results confirms:1,from the levels of molecular Polysaccharide,we confirm that the space environment have the mutagenic effection on the Streptococcus;2,Experiments prove that the space environment can selective affect the expression of polysaccharide in streptococcus. the levels of the expression of the ConA related polysaccharide with High Mannose,SNA related terminal Neu5Aca2-6Gal and RCA-Ⅰrelated Galβ1-4GlcNAc are up and the levels of the expression of the WGA related polysaccharide with terminal GlcNAc and PHA related terminal Gal-Nac have no significant changes;3,According to the effection of the expression of the bacterial polysaccharide,we can use a new way for polysaccharide biological products screening and enhancing performance or improving production and we can possible provide a new experiental evidence of the biological functions for the space mutation bacteria;4,The mechanisms of regulation to the expressions of Streptococcus polysaccharide molecular in space environment have to be further studied.
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