Isolation, Purification And Functional Analysis Of LP7, One Member Of 30K Family In Silkworm, Bombyx Mori.

30K proteins,a family of low molecular lipoprotein,express highly during fifth instar larvae of silkworm,which are specific lipoprotein in silkworm.They are synthesized in the fat body then secreted to hemolymph.30K proteins are not only the main storage protein for growth,but also the important energy material of the development of embryo.Till now,the function about 30K protein is scarce and the function about physiological is not clear completely.30K proteins have multi-members,whose comparability is so high that is difficult to separate and purify.At present,4 members of 30K proteins have been separated and purified sucessfully.How many kinds of 30K protein need to further study.After the complete of silkworm genome,we have predicted 10 genes of 30K family by utilizing the database.By analysis of the data,we found that the amino acid numbers encoded by those genes are around 246-271.The theoretical pI of them are between 6.1-8.4 and the molecular weight of them are between 28kD-31kD.What's more,the similarity of the amino acid sequences is above 60%.These information offered important reference for research of 30K protein.Based on the result,we started purification of 30K protein,obtained a novel protein,and carried on further analysis and research.Considering the difference of the molecular weight and theoretical pI of the 30K proteins,we have designed the purified experiment procedure and experiment condition of separating.By Ammonium sulfate precipitation,and three separate technologies(cation ion-exchange chromatography,anion ion.exchange chromatography and hydrophobic chromatography),we separate the proteins of the hemolymph of silkworm sucessfully and got a single strip of 30K protein.By analysis using Assist matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),the single signal peak of this protein sample is obtained.It was proved that we had been succeeded in getting a single member of 30K protein in this research.Meanwhile,we got the PMF(peptide mass fingerprint map)of this protein and utilized GPMAW(General Protein/Mass Analysis for Windows) software to analyze in the silkworm protein database.We found that this protein is encoded by 30K gene BmlpT.Compared the amino acid sequences of this protein with 30K protein reported,We found this protein is a new protein of 30K protein LP7.Through polyclone antibody preparation,we obtained the antibody of LP7.Firstly,we regarded 30K protein LP7 as the antigen and injected it to the rabbit and carried on the immune program. Then,gathered the blood from the arterial,thus succeeded in getting antiserum.Utilized the saturation solution of ammonium sulfate to purify the antibody in antiserum,we removed a large number of impurity and other protein.So we succeed in getting the antibody of 30K protein LP7 which can meet the requirement of next experiment.By Western blot,we have investigated the 30K protein LP7 in every organization of some periods of silkworm.The result showed that in the 3^thday of the fifth instar larvae,LP7 was detected in the hemolymph,head,fat body and gonad,and the level in the hemolymph is the highest.in addition, LP7 appeared faintly in skin and midgut,but never detected in the silk gland.At the same time,we analyzed the Gene Chip,and the result showed that gene Bmlp7 was expressed in head,fat body, skin and gonad,So,we deduce that LP7 in hemolymph is shifed from fat body,and LP7 in midgut may be shifod from hemolymph.The expression amount of Bmlp7 gene is relatively low in silk gland and malpighian tubule,so wo can't detect the protein.In the 3^thday of the moth,we detected LP7 in all organization almost,but the expression level of LP7 is faint in the hemolymph,while highest in the gonad.The pattern of 30K protein in silkworm,that suggested its important function in growth of the silkworm,especially in growth of the embryo.At the same time,by western blot,we had investigated the hemolymph and gonad of some periods of the silkworm.The result showed,in the hemolymph,LP7 was detected since the 3^thday of five instar,and the signal level of LP7 increased gradually,during the first day of pupa,it reach peak, then reduce gradually and while in the 3^thday of moth the signal is very faint;In the gonad,the circs of LP7 reverse completely.In the 3^thday of fifth instar,its signal is very faint,and then increase gradually until the moth when reached the peak.So this protein is important to development of silkworm's embryo.It may be the important energy substance.We also had investigated the male and female gonad of the 6^thday of pupa,LP7 was detected in male and female.It suggested LP7 protein have important function in procreant growth.At the same time,we analyzed the Gene Chip,and the result showed that gene Bmlp7 was expressed since the anaphase of five instar,and the signal level increased gradually,during the prophase of pupa,it reach peak,then reduced,and increased in moth. But existing research indicates expression of this gene in the fat body only last to the 5^thday of pupa. It is concluded that LP7 was shifted from the hemolymph to gonad,the gonad oneself might format this protein at the same time.Furthermore,using the glass capillary syringe,Aspergillus niger,a kind of fungi,was injected into the body of the 3^thday of pupa,the hemolymph of 24h and 48h were collected for analysis of LP7 in the hemolymph by Western blot.The result showed that in the injected pupa,the LP7 show a faint increase,compare to the control in the normal situation where the amount of LP7 in hemolymph should be reduced gradually after the 3^thday of pupa.after injected,it maybe promoted the synthesizing of LP7,also maybe have some reasons to hold back the shifted of LP7,induced LP7 protein increased in hemolymph.So,we deduce that LP7 may be related to silkworm's immune system.As a result,we purified successfully a novel protein,a member of 30K protein family from the hemolymph of silkworm,named as LP7.After preparation of polyclone antibody of this protein,and the result of Gene Chip,we carried on the preliminary discussion about its possible function.It made a fundament for further studying of LP7.It was also offered important data for researching 30K protein family.At the same time,it offered the new theoretical entry in researching the immune mechanism of silkworm.