Study On The Cloning Expression, Purification And Anti-tumor Activities Of Gene Latcripin-15 Functional Domain

Objective: In order to seek the sole functional protein with high anti-tumor activity, apoptosis-related gene Latcripin-15 from Lentinula edodes C91-3 transcriptome was found and its gene sequences and functional groups were identified. By building p ET32a(+)-LP15-RCC1 prokaryotic expression vector, apoptosis-related LP15-RCC1 protein was induced expression in E.coli Rosetta-gami(DE3) and identified its expression product. A preliminary study on the influence of apoptosis related LP15-RCC1 protein from Lentinula edodes C91-3 in different tumor proliferation activity. While induced apoptosis in human lung cancer cell A549 by LP15-RCC1 protein was investigated specifically. These researches laid a foundation for further study of the mechanism of inducing apoptosis of tumor cells.Methods :(1)Total RNA was extracted from the mycelium of Lentinula edodes C91–3。The full- length gene of Latcripin-15 was isolated with 3’-Full Rapid Amplification of c DN A Ends(RACE) and 5’-Full RAC E methods according to the Lentinula edodes transcriptome sequencing results. Latcripin-15 gene domain was obtained through comparative analysis of Pfam database. Specific primers were designed to get Latcripin-15 domain sequences by using RCR technology. Amino acid composition, physicochemical properties and structural domain of the protein were analyzed with bioinformatics software.(2) The Latcripin-15 domain gene was cloned into the prokaryotic expression vector p ET32a(+) to construct recombinant plasmid p ET32a(+)-LP15-RCC1. The plasmid p ET32a(+)-LP15-RCC1 was transformed into E.coli Rosetta-gami(DE3) to inducible expression. The expression products were identified by SDS-PAGE and Western blot. Using affinity chromatography for target protein separation and purification and the protein concentration was measured with BCA method.(3)The anti- tumor activity of LP15-RCC1 protein was identified. After treated with LP15-RCC1 protein, the growth inhibition rate of human tumor cells(lung cancer A549 cells, cervical cancer Hela cells and hepatoma Hep G2 cells) was detected by MTT(Methyl Thiazoly Ltetra Zolium, MTT) assay and the morphology impact of human lung cancer A549 cells was observed by transmission electron microscopy. Cells Apoptosis of human lung cancer A549 was studied using flow cytometry and Hoechst33258 staining.(4)Using MTT method to detect LP15-RCC1 protein on the normal chicken embryos into the toxic effects of fiber cells.Results:(1)Latcripin-15 full- length gene was obtained by RACE method and Latcripin-15 domain fragment sequences was got by PCR. Through the analysis of Latcripin-15 full- length amino acid sequence, we found that the protein contain a plurality of chromosome condensation regulator domain(RCC1).(2)We could determine that the fragment which inserted into the p ET32a(+) was the Latcripin-15 domain gene fragments by double digestion with Eco RⅠand XhoⅠ. SDS-PAGE and Western blot results showed that the protein expressed in Ecoli. Rosetta- gami(DE3) was the LP15-RCC1 protein. Purified single target protein was got successfully by affinity chromatography column. We used BCA method to measure the concentration of LP15-RCC1 domain protein. The result showed that standard linear equation is y = 0.0628 x + 0.0012, R2 = 0.999. According to equation, the concentration of LP15-RCC1 domain protein is 1.74 mg/ml.(3)MTT results showed that different concentrations of LP15-RCC1 protein could inhibit the growth of human tumor cells(lung cancer A549 cells, cervical cancer Hela cells and hepatoma Hep G2 cells) significantly compared with the control group, and the difference was significant(P<0.05). Cell apoptosis was found in human lung cancer A549 cells after treated with 200 μg/ml LP15-RCC1 protein for 48 h through TEM observed, while cell structure of the control group cells remained normal. Flow cytometry results revealed that LP15-RCC1 protein has the ability of inducing human lung cancer A549 cells to early apoptosis. When the concentration of the protein is 200 μg/ml, the apoptosis inducing capacity is the best. The early apoptosis of the A549 cells has a significant difference between the experimental group and control group(P<0.05). Hoechst33258 staining results showed that apoptotic A549 cells have been observed through fluorescence microscopy in 50μg/ml and 100 μg/ml LP15-RCC1 protein group, while the control group was not found apoptotic A549 cells.(4)MTT results indicated that there is no toxic effect for normal chick embryo fibroblast cells.Conclusion:(1) Apoptosis-related gene Latcripin-15 was screened out from Lentinula edodes C91-3 transcriptome according to the transcriptome sequences. Latcripin-15 full- length gene and Latcripin-15 domain fragment sequences was obtained successfully.(2) Recombinant plasmid p ET32a(+)-LP15-RCC1 has been constructed successfully. The apoptosis-related LP15-RCC1 protein has been successfully expressed in E.coli Rosetta-gami(DE3). LP15-RCC1 protein was successfully purified by affinity chromatography. LP15-RCC1 protein was refolded by urea gradient dialysis.(3)LP15-RCC1 protein has growth inhibitory effects on human lung cancer A549 cells, human Hela cells and human hepatoma Hep G2 cells. At the same time, the protein had no toxic effect on normal chick embryo fibroblast cells. Preliminary studies have shown that LP15-RCC1 protein can induce apoptosis on human lung cancer A549 cells. But the specific mechanism needs to be studied in the furture. This experiment lays a compacted foundation for further study about anti-tumor mechanism of Lentinula edodes C91-3.