The Preparation Of Polyclonal Antibody Against Trichinella Spiralis ESF1Protein And Its Influence On Human Colon Cancer HCT-8Cells

Colon cancer is a common malignant tumor in gastrointestinal tumors, and it isthe third most common cancers in men and the second in women, the second leadingcause of cancer related death. During the past20years, there has been an increasingincidence especially in the big cities of China. The symptoms of early colon cancerare not obvious, so most patients with stage â…¡/â…¢ are diagnosed. The only surgicaltreatment of colon cancer cannot effectively improve the prognosis of patients, so thecomprehensive therapy has become an important therapeutic principle. Biotherapy, asa part of the comprehensive treatment, combined with surgery and adjuvantchemotherapy, has become one of research hotpots because of its advantages, such ashigh curative effect, strong specificity, and relative security.Our laboratory selected gene Tsp₀9347, which is related with HCT–8cells,from Trichinella spiralis by constructing cDNA expression library.Tsp₀9347encodesa protein called ESF1. There was no report about Trichinella spiralis ESF1protein, sothe biological functions of this protein were unknown. There have been report aboutthat Trichinella spiralis can resist tumor. The previous studies suggested Trichinellaspiralis inhibited the growth of SP2/0myeloma cells, A549lung cancer cells, HCT-8colon cancer cells and Hepal–6hepatic carcinoma cells. The live Thrichinellaspiralis have a potential risk to host, therefore, in this study，we expressed Trichinellaspiralis ESF1protein by constructing prokaryotic expression vector, producedpolyclonal antibody against ESF1protein from BABL/c mice, and observed theinhibitory effect on HCT-8cells. Our study may provide a new approach for thebiotherapy of the colon cancer. Methods:1. The construction of Trichinella spiralis gene Tsp₀9347prokaryoticexpression vector: we obtained Tsp₀9347length gene by RT-PCR, and connected itto pMD-18T vector, and then constructed recombinant plasmidpET-28a（+）-Tsp₀9347. These results were identified by seqμencing.2. The identification and purification of ESF1protein: Recombinant plasmidpET-28a（+）-Tsp₀9347was transformed into E.coli, and was induced to expresstargeted protein, which was identified by SDS-PAGE and western blotting, and thenwas purified from inclusion bodies.3.The preparation of polyclonal antibody against ESF1protein: BALB/c micewere injected with ESF1protein subcutaneously, and the titer of the antiserum wasdetected by indirect ELISA.4. The inhibitory effect of polyclonal antibody against ESF1on HCT-8cells wasobserved by MTT in vitro.5. The observation of the influence of polyclonal antibody against ESF1proteinon human colon carcinoma implant tumor in nude mice: the effect of polyclonalantibody against ESF1protein on the growth rate of tumor by constructing HCT-8human colon cancer cells implanted subcutaneously in nude mice.Results:1. The results of restriction enzyme digestion and sequencing are consistent withthe mRNA of Trichinella spiralis ESF1protein from NCBI.2. Western blotting showed that the expressed protein is the Trichinella spiralisESF1protein.3. The titer of polyclonal antibody against ESF1protein is1:204800which wasdetected by indirect ELISA4. MTT result manifested that, contrasted to control group, the polyclonalantibody against ESF1inhibited the growth of HCT-8cells obviously （P＜0.05）.5. The tumors’ volumes of the polyclonal antibody against ESF1protein groupand the positive control group are smaller than control group’s（P＜0.05）, and thetumors’ volumes of the polyclonal antibody against ESF1protein group are smaller than the positive control group’s.Conclusions:1. The Trichinella spiralis ESF1protein, which was expressed by constructingthe prokaryotic expression vector, was identified by western blotting. We prepared thepolyclonal antibody against ESF1, and its titer is μp to1:204800.2. The polyclonal antibody against ESF1which is encoded by Trichinella spiralisgene Tsp₀9347had obviously inhibitory effect on colon cancer cell line HCT-8invitro.3. The polyclonal antibody against ESF1significantly inhibited the growth ofHCT-8cell tumor in mice, and the maximum inhibition rate is to45.50%.