The Expression Study Of BRDT-NY Protein

In the past many years, there has been a continuing search for cancer vaccine targets. Owing to their highly tissue-restricted expression in the testis and the various malignancies, CT antigens are being vigorously pursued as targets for therapeutic cancer vaccines.BRDT-NY gene is a novel alternative splice variant of CT9 (also named BRDT gene). It is also a kind of Bromodomain gene. Multiple tissue RT-PCR results showed that BRDT-NY gene only expressed in testis, with no expression in other normal human tissues. Till now, the protein expression of BRDT-NY has not been reported.The aims of this study are: First to express and purify recombinant BRDT-NY proteins, then these proteins were used as immunogens, with which a high-titer anti-BRDT-NY polyclonal antibody was successfully obtained by immunization of BalB/c mice; Second to detect the expression of BRDT-NY protein in human normal testis; Third to study the expression of BRDT-NY protein in different malignant tumors. The research divides into following three parts:Part 1. Cloning and expressing the fragments of BRDT-NY gene, and purifying the BRDT-NY recombinant proteinAccording to BRDT-NY nuclear acid sequences,three primer pairs were designed. Three fragments of BRDT-NY gene, named BRDT-NY₁, BRDT-NY₂, BRDT-NY₃. BRDT-NY₁, BRDT-NY₂, BRDT-NY₃, were amplified from pTriplEx2-BRDT-NY by PCR respectively. Then the three fragments were inserted into pET_28a+ vector. These recombinant vectors were transformed into E.coli BL21 and induced by IPTG for expressing 6×His-tag fusion protein BRDT-NY₁, BRDT-NY₂, BRDT-NY₃. The proteins were purified and annealed.Part 2. Preparing anti-BRDT-NY polyclonal antibodyThe protein purfied with Ni-NTA were used as antigens to immunize eight months old BALB/c mice for four times. ELISA analysis showed antiserum had high affinity. The results of Western-blot demonstrated that only AbBRDT-NY1 could specifically recognize endogenous BRDT-NY protein in testis, so it can be used in the following immunohistochemistry experiments.Part 3. Expression study of BRDT-NY protein in human testis and different malignant tumorsTo determine in which types of cells in the testis BRDT-NY protein were expressed, immunohistochemical analysis was carried out using sections of human testis. BRDT-NY protein was detected primarily in the nucleolus of round spermatidsm. Little signal or no signal was detected in the other spermatogenic cells. These results suggest that BRDT-NY may have a role during spermiogenesis.We also examined the expression profile of BRDT-NY protein in human different malignant tumors (44 cases). The results showed that BRDT-NY protein was detected in 11 of 14 cases of esophageal cancer and 2 of 5 cases non-tumorous esophageal tissues. All 10 cases of gastric cancer expressed BRDT-NY protein. While 2 of 10 cases non-tumorous gastric tissues also had BRDT-NY protein signal. The result had significant difference in statistics (P<0.05).These results suggested that the incidence of esophageal and gastric tumors was higher than the non-tumorous tissues. BRDT-NY protein may be used as a target for diagnosis and therapy of esophageal and gastric cancer.Among 10 cases of breast cancer, 8 cases showed positive signal of BRDT-NY protein. However, in non-tumorous breast tissues the positive rate was also high (5/7). But the distribution of the BRDT-NY protein was different in breast cancer and non-tumorous breast tissues. To determine the significance of BRDT-NY protein expression in breast cancer, more cases will need.All of the 5 cases of colon cancer, three cases of thyroid cancer, one case of lung cancer and one case of ovarian cancer, BRDT-NY protein expression were positive. And almost none of the non-tumorous tissues were positive. Further studies with larger sample size may be required to analysis the possibility of BRDT-NY as a target for therapeutic cancer vaccine. In conclusion, BRDT-NY might be used as targets to develop safe and effective gene-based therapy for malignant tumors, especially for esophageal and gastric cancer.