The Purification, Gene Cloning And Activity Analysis Of A New Protein Elicitor From Alternaria Tenuissima

A protein elicitor from Alternaria tenuissima,which was stored in our lab,was sepearated and purified and then the protein's gene was cloned using RT-PCR and RACE techologly.The gene was connected to the express vectors and expressed in E.coli and purified by the chelating column.The purification and gene clone of the protein set up a basis for us to research the mechanism of the protein to plants.The total results were showed as follow:1.The culture of the Alternaria tenuissima and the purification of the proteinThe Alternaria tenuissima was cultured in PD medium,and then Tris-HCl buffer extracted the total proteins.The total proteins were concentrated by ammonium sulphate fractionation,cold alcohol precipitation,and then purified a certain elicitor by anion exchange chromatography and size exclusion chromatography.The result showed that the MES buffer at the pH6.0,which contained 0.4M NaCl,could purify the elicitor.The elution of the single peak after size exclusion chromatography showed a single band on the SDS-PAGE.The pI=4.22of the purified Plant elicitor was acquired by 2-DE techologly.2.The purification of the antibody against the protein using affinity chromatography methodThe special antibody against the protein was purified successfully from the rabbit blood serum.The Western-Blot result showed that the antibody could bind specially to the protein,and the concentration equal to 1/10 of the crude blood serum.3.The gene clone of the elicitorThe cDNA was acquired from total RNA of Alternaria tenuissima using reverse translation method;then RT-PCR was operated using couples of primers based on the sequences acquired by MALDI-TOF,MALDI-TOF/TOF,microLC-ESI,nano LC-ESI-IT-MS/MS methods,the sequences are showed as follow:1.LFVINKPDVYKSPSSNTWIIFGEAK2.DIELVMQQASVSR3.ALKENDNDIVNSIMALSI4.RPKNILFVINQPDVYKA middle gene sequence,which has a length of 373bp,was got by several times of RT-PCR actions.The full-length of the cDNA gene,which has a length of 624bp,was acquired by 3' and 5'RACE methods.The gene encodes a protein which has 207 amino acids,and its logical MW is 22.590kD,logical pI is 4.43,the gene was enrolled in GenBank,the enroll No.is CH445335.We also cloned the DNA gene using PCR method,the result showed that the DNA gene has 2 intrones between 237～350,409～513bps.4.The expression of the recombinant protein and bioassay analysisThe full-length eDNA sequence of the gene was connected to the expression vectors pET-28a and pGEX-6P-1 and expressed in E.coli separately.The recombinant protein with a His-tag was purified by chelating.The MS detection showed that the recombinant protein has a same molecular weight as its logical molecular weight,it is clear that the protein has an abnormal electrophoresis phenomenon.Then the bioassay of the purified and recombinant proteins were detected,the results showed that they have the same function in improving the activity of succinate dehydrogenase.