Studies Of Antiviral Protein From Stellaria Media L

Stellaria media (L.) Villars is a medicinal and edible plant which belongs to Stellaria L. Caryophyllaceae. S. media can be used as medicine and its fresh juice has remarkable efficacy of curing some virus skin diseases, such as HSV. In this paper, in order to clarify its effective substances, a unique anti-HSV-targeted extraction technique was used to isolate a new bioactive protein from S. media. We have analyzed its primary structure, physicochemical properties and biologic activities, and found that it has the activities of peroxidase (POD) and ribosome-inactivating protein (RIP). Meanwhile, we have studied the homologous cloning and heterologous expression of RIP, and succeeded in cloning two new RIP genes Q1, Q2and getting its heterologous expression. It has provided important information for purification and clarification of the antivirual effective substances from S. media. The following is the main research results:1. Isolation, purification and identification of bioactive proteins from S. media and the studies of its biologic activity.1.1Isolation and purification of bioactive proteins from S. mediaBioactive protein was purified from S. media by50%-85%ammonia sulfate precipitation, CM Sepharose Fast Flow cation-exchange chromatography, Sephadex G-75gel filtration chromatography and HiTrap SP HP cation-exchange chromatography in this study. By SDS-PAGE、FPLC、MALDI-TOF MS, the protein was at least chromatographic pure. Its accurate molecular weight is35.157kD. It was proved to be a basic protein with pI about pH9.5by IEF-PAGE, and a glycoprotein by PAS.1.2Identification of bioactive proteins from S. mediaMALDI-TOF MS was applied to the analysis of peptide mass fingerprinting (PMF) in bioactive protein. The PMF data was used in Mascot search, but no data matched. ESI-Q-TOF2MS was used in bioactive protein identification. Four tryptic-digested peptide fragments were sequenced by ESI-MS/MS, and the sequences were:GFEVIDAAK (Frl), GPSFAVQLR (Fr2), AFSTNKGLAPGLLR (Fr3) and MGNTGVLTGERNDR (Fr4). No data matched in database search as well. Multiple alignments with POD sequences showed there are some conserved amino acids in Frl and Fr4. So the bioactive protein is a new natural plant protein, which named SAP (Stellaria Antiviral Protein). 1.3The studies of biologic activity of bioactive proteins from S. mediaAs shown in the experiment, the SAP has anti-virus and anti-tumor activity. The anti-virus in vitro experiment shows that SAP can inhibit HSV-2with an IC50value of0.37μM and CC50>40μM. SAP affect the initial stage of HSV-2infection, and the mechanism relates to inhibit virus bioactive. As shown in the anti-tumor in vitro experiment, SAP has a strong inhibitory activity on HL-60and LoVo with IC50of9.09μM and12.32μM respectively, while it has no obvious effect on the normal cells, with CC50>40μM. So it has selective inhibition on the tumor cells.SAP has strong N-activity on28S rRNA and DNA-cleaving activity on supercoiled DNA. So SAP is a kind of DNA (RNA) glycosidase/depurination cleavage enzyme as it can effect on the RNA and DNA cleavage beyond retrieve. This kind of enzyme activity may be one of mechanism of anti-virus and anti-tumor effects. The POD activity in vitro experiment shows that Stellarmdin A has obvious peroxidase activity with enzyme activity of36.6Umin-1μg-1and Km of0.01mol/L.2. Cloning and prokaryotic expression of ribosome-inactivating protein (RIP) Genes from S. media2.1Cloning and analysis of RIP Genes from S. mediaThe method of homologous cloning was applied to obtain the conserved fragments of gene encoding RIP from S. media. Combined with rapid amplification of cDNA ends (RACE),5’and3’ends were got. Linked the fragments,2genes encoding RIPs were obtained. Designed primers at initial and terminal codon and used cDNA as template, Q1(FJ860049) and Q2(FJ860050) were acquired. The length of open reading fragment (ORF) of Q1was858bp, and that of Q2was765bp. The similarity between Q1and Q2was95.5%. There were390base pairs which are the same as other RIP genes in Caryophyllaceae and distributed orderly. But there was no similarity between S. media and the plant of other family.The proteins encoded by Q1and Q2were named Stellarin1and Stellarin2. Their putative molecular weights were31.0kD and27.6kD, and pI were9.40and9.54. Furthermore, the two proteins contained signal peptide composed by23amino acids. Their physicochemical property has similarities with I type RIP and SAP, although it has lower homology with the latter. The Sequence shows that Stellarin1and Stellarin2has high homology with RIP anti-virus activity characteristics area and has completely same conservative areas. This proves that the RIP of S. media has antivirual ability, which indicates that S. media has many kinds of antivirual protein.2.2Prokaryotic expression of RIP Genes from S. mediaConnect Q1and Q2to pET29a to construct expression vector:pET29a-Q1and pET29a-Q2. Then the vectors were transformed to E. coli strains, such as DH5a, BL21(DE3) and BL21(DE3) plysS, cultivated the E. coli strains in LB and TB medium and induced expression of Q1and Q2. But SDS-PAGE showed that Q1didn’t express in any strains and culture medium. Q2expressed in BL21(DE3) and BL21(DE3) plysS in LB, but only a little. Maybe the products of Q1and Q2were too poisonous for E. coli.