The Expression And Functional Study Of LP1-PI3K Domain From Lentinula Edodes C91-3

Objective:This study firstly aimed to construct a prokaryotic expressing vector of LP1-PI3K(latcripin-1-PI3K) gene from Latcripin-1in Lentinula edodes C91-3. Then the proteinof LP1-PI3K was expressed and purified. Finally, the anti-tumor biological function ofprotein LP1-PI3K on A549cells was analyzed and confirmed.Method:1. According to the gene sequence of LP1from GenBank: JQ327159.1, a pair ofspecific primers to amplify the target genes LP1-PI3K was designed.Then it was clonedinto the cloning vector—pMD19-T Simple Vector and was selected by the blue andwhite spot experiment. Next, the LP1-PI3K gene was selected by restriction enzymedigestion and cloned into the expressing vector-pET-32a.2. To express the protein, the plasmid pET-32a-LP1-PI3K was transformed intocompetent E.coli Rosetta-gami (DE3). The objective protein was over expressed withisopropythio-beta-D-galctoside (IPTG) and purified by Ni-NTA agarose.3. Flow cytometry and3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazoliumBromide (MTT) methods were used to detect the biological results of domain PI3K tolung cancer cell. Transmission electron microscopy (TEM) technology was used toanalyze the ultrastructure of the treated cell. Western blot was used to identify thefunctional domain and the autophagosomal marker—Microtuble associated protein lightchain3(LC3).Result:1. The LP1-PI3K gene clone was checked by PCR, double enzymatic digestion andgene sequence. Compared with the original genes, there was no change on the level ofamino acid. 2.12%Sodium Dodecyl sulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE)analysis and Western-blot showed that the molecular weight of target protein is about44KDa.12%SDS-PAGE and western blotting also revealed that the protein wasexpressed efficiently and constantly in E.coli Rosetta-gami (DE3) cells. Then proteinLP1-PI3K was carefully purified by6×His Tagged Ni-NTA affinity chromatography.3．The biological function analysis:①The MTT results showed the growth ofcancer cells A549was inhibited significantly by this functional domain. With proteinconcentration of15μg/ml,30μg/ml,60μg/ml and at the time of24,48,72h we used toA549. It is demonstrated that the LP1-PI3K domain inhibit the growth of A549cells ina time/dose-dependent manner. There was statistically significant differences betweenthe control group and experimental group (P﹤0.05).②By flow cytometer examination,with the protein concentration of30μg/ml,60μg/ml and120μg/ml after48h, the earlyand advanced apoptosis rate of target protein were8.49%,18.58%,20.45%and5.01%,10.06%,14.88%correspondingly. By Cell cycle analysis, in the experimental group, theapoptotic peak appeared before the G1phase peak. But there is no apparent dosedependence.③In the control group, A549cells maintained regular morphology instructural integrity with a large nucleus, some mitochondria and membrane phase.However, after A549cells were incubated with LP1-PI3K functional domain for48hours, many chromatin condensation, nuclear fragmentation, intracytoplasmic vacuolesand especially a lot of autophagosome could be seen.④In the control group, the twotypes of LC3were both lower, while in the experimental group, the expression ofLC3-II was significantly higher than that of LC3-I.Conclusion:1. With the designed specific primer, the sequence of LP1-PI3K function domainwas obtained by PCR. The recombinant plasmid pET-32a-LP1-PI3K was alsoconstructed.2. The LP1-PI3K functional domain was expressed with Rosetta gami prokaryoticexpression and purification system. The expressed protein was purified with His-tagaffinity chromatography and renatured in urea.3. The biological function analysis showed that it could enhance the appearance ofautophagosome in A549cells and could also inhibit the growth of A549cells.It may beinvolved in regulation of the growth of tumor cells through autophagy pathway. TheLP1-PI3K functional domain could induce autophagy to programmed cell death. Thisresearch on this functional domain of LP1is of significant value for the development of new polypeptide drugs. All these lay a foundation for further researches onLP1-PI3K s anti-tumor biological functions and mechanism.