Study On The Role Of Eupolyphaga Fibrinolytic Protein To S180Cell By Gene Cloning Technology And Transfection Method

Eupolyphaga is a medicinal herbal with a long history, and recent studies have found that it is abundant in protein and other nutrients. The advantages of eupolyphaga including strong viability, fast growth, strong adaptability, breeding with low cost etc. make the breeding of it become one of the fastest growing breeding insects industry. Blood circulation effect of the eupolyphaga is closely related to a fibrinolytic protein, and vitro experiments have high thrombolytic performance through the separation and extraction of proteins. Further stud found that eupolyphagafibrinolytic protein has a strong anti-tumor effect, but the mechanism is not very clear. Currently cancer and cardiovascular disease are the two major life killers in the world. The author hopes that further stud} of the active protein gene providing a more complete basic experimental data can lay the foundation to find an efficient, safe and low-cost drug in the future.This experiment designs appropriate primers by referring to obtained the fibrinolytic protein genes of eupolyphaga and suitable selected restriction sites of cloning sites carrier from laboratories and also reverse transcriptase first-strand cDNA. retrieval the fibrinolytic of eupolyphaga protease through PCR by the extraction of total RNA turtle. And then by the TA cloning connect fibrinolytic genes to the PM19. select positive clones by blue and white screening and do PCR validation. Extract fibrinolytic protein gene fragment by double digestion to build s180cells pCDNA3.1(+)-EFP transfection. Observe and record cell morphology while screening expressing lineage cells under the pressure of G418. Extract the cell proteins of stable expression cells and do SDS-PAGE analyze expression of the intracellular protein. The experiments found that transfected pcDNA3.1(+)-EFP plasmid vector of s180cells changes from spindle to round and cell adhesion decreases and adherence is not strong. There appears flocculent grape string-like cell clusters in the cell culture medium. The fibrinolytic protein of eupolyphaga can be expressed in s180cells through detection, leading to the above phenomenon. It turns out that the fibrinolytic protein of Eupolyphaga have such effects as anti-tumor cell adhesion invasion. metastasis etc. It also shows that the fibrinolytic protein gene of Eupolyphaga can be used to build the carrier of the anti-tumor cancer gene therapy. However, there is a lot of work to do because of unclear anti-tumor mechanism of the fibrinoltic protein of Eupolyphaga.