Preliminary Study On Hemolytic Activity Of Recombinant Expression Product Of Tlh In Vibrio Parahaemolyticus And Inhibition Of Proliferation Of KYSE450 Cells

ObjectiveVibrio parahaemolyticus(VP)is present in temperate and tropical marine systems,and food poisoning usually occurs in the summer,mainly related to the consumption of raw or undercooked seafood.The typical clinical symptom of VP is acute gastroenteritis,often accompanied by abdominal cramps,diarrhea,nausea,vomiting,fever,chills and watery stools.In China’s coastal areas,food poisoning caused by VP is at the forefront.The tlh gene encodes Thermomolbine Hemolysin(TLH),which is a targeted gene in the genetic detection process of VP,but the pathogenesis of TLH is not yet clear.For the first time,Pichia pastoris was used to construct the exogenous protein expression system of VP thermomolbine hemolysin,which was expressed and secreted,its molecular weight and hemolysis function were verified,and investigate the inhibitory effect of TLH on the proliferation of human esophageal cancer cell line KYSE450,providing valuable reference for cancer cell proliferation inhibition research and anticancer drug development.MethodsThe strain was activated on the basis that the recombinant expression plasmid pPIC9K-tlh was successfully transformed into the complete strain of Pichia pastoris SMD1168 by electroporation.A high-copy positive strain was selected,and the target protein(TLH)was secreted in a methanol-containing induction medium.The secreted supernatant was centrifuged,and the target protein was isolated by trichloroacetic acid(TCA)-acetone precipitation.The protein size was determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE),and lecithin was added to detect the hemolysis function of the target protein.Finally,the dose-response curve of the inhibition of human esophageal cancer cell line KYSE450 by TLH was determined using the Cell Counting Kit-8(CCK-8).Results(1)The results of SDS-PAGE showed that the molecular weight of the secreted protein was close to 43 KD,which was consistent with the references,which proved that the target protein was successfully expressed.(2)The lecithin was added to the target protein secreted by the engineering strain,and the hemolysis test result was positive,while the control group was negative,indicating that the target protein has a lecithin-dependent hemolytic activity,which is consistent with the function reported by the TLH,and the target protein was verified again.(3)The activity of human esophageal cancer cell KYSE450 decreased with the increase of target protein concentration,and it was presumed that TLH inhibited the proliferation of human esophageal cancer cell line KYSE450.ConclusionThe TLH was successfully expressed by the Pichia pastoris host secreting and expressing the extracellular protein system.The hemolytic function was confirmed by experiments,indicating that the target protein has phosphatase-dependent hemolytic activity,and the degree of hemolysis increases as the induction time of the engineered strain increases.The CCK-8 experiment proved that it inhibits on human esophageal cancer cells.KYSE450 proliferation.