Biopanning The Interaction Protein With Cholangiocarcinoma Cells From Adult Of Clonorchis Sinensis

The Oriental liver fluker, C. sinensis, is a serious fish-borne parasites, not only caused the huge economic losses to fishery production, but also made a great threat to human's health. These flukes are predominantly endemic in the East and Southeast Asia countries, such as China, Korea, Vietnam and Laos, and is the highly prevalent in China. According to the epidemiological survey, C. sinensis has been reported for most provinces in China, except Inner Mongolia, Ningxia, Qinghai, Tibet, and Xinjiang, and over 15 million people are infected with C. sinensis in our country now, especially in Guangdong, Guangxi and the three northeast provinces. Clonorchiasis is a serious food-borne parasitic zoonosis, which is mainly exhibited as hepatobiliary lesions caused by parasitic C. sinensis in human bile ducts. The liver appears grossly normal in light infections, but in heavy infections there is the enlargement of the liver and loss of appetite with acute pain in the right upper quadrant. In the chronic stages, the liver might eventually become malfunctioning. Biliary obstruction, intrahepatic stone formation, cholangitis, cholecystitis, cholelithiasis, biliary and liver abscesses, and pancreatitis are further severe symptoms. In recent years, both experimental and epidemiologic evidence strongly implicates C. sinensis infection in the aetiology of one of the liver cancer subtypes—cholangiocarcinoma (CCA), or cancer of the bile ducts. Like O. viverrini, C. sinensis is also classified as Group 1 carcinogenic to humans by the International Agency for Research on Cancer for cholangiocarcinoma in 2009.The exact mechanisms of the carcinogenesis, however, are not clearly elucidated. The mechanical damage, parasite secretions and immunopathology may enhance cholangiocarcinogenesis. The ?uke excretory–secretory several metabolic products (ES), which was released from the tegument and excretory opening of C. sinensis during host infection, apart from inducing host immune response, may be toxic to or interact with biliary epithelium. In order to further identify that ES from the liver ?ukes play pivotal roles in development of cholangiocarcinoma, we collected the amounts of ES products of C. sinensis, and treated the cholangiocarcinoma cells lines and hepatocellular carcinoma cell lines in different time and in different concentration. We used the CCK-8 method to measure cell proliferation, and the result showed that cells treated with ES products significantly promote the proliferation of two cells compared to control cells. These findings support a role for ES products of C. sinensis in establishment of a tumorigenic environment.The phage display technique has been widely used in many research areas of the life sciences, such as the reaction of antigen-antibody, interactions of protein-protein and protein-drug, because it has many advantages, for example high library capacity, screening of convenient and flexible. In order to biopan the interaction protein with cholangiocarcinoma cells from adult of C. sinensis, we constructed successfully a T7 phage display cDNA library of adult worms, and the primitive library capacity was 3.7×105pfu, recombinant ratio was 98%, and the title of amplification library was 4.3×1012pfu/ml. Then, the display library was screened by whole-cell selection method. The phages binding on the cells were stable after 5 turn's biopanning. Finally, we obtained 6 proteins which can interact with cholangiocarcinoma cells by sequencing and analyzing, and separately named Cs-CCA-p8,4,13,21,7,14. They are highly homologous with C. sinensis, S. mansoni and S. japonicum, therefore, this implies that they may have similar biological functions. There were not transmembrane domains in the six proteins through TMHMM Analyzing, however, Cs-CCA-p4 and Cs-CCA-p7 contain a signal peptide, therefore, they may be transported through the way of secretion to play a role in extracellular. There provide a more comprehensive solution to obtain the carcinogenic-related genes by biopanning of display library combination with other screening methods, for example the electronic screening of EST sequence.