本實驗室過去已應用基因重組的技術，以穀胱轉移基因接合系統（glutathione S-transferase gene fusion system）將X基因嵌入質體pGEX-5x-1中， 再將此重組質體植入大腸桿菌。本實驗則以免疫分析法對所合成的重組蛋白質進行定性分析。重組質體的核酸序列分析，證實了X基因已正確地（in frame）嵌入pGEX-5x-1質體中，接著進行表達、純化GST-X接合蛋白質，在純化的過程中發現，置換後的醣珠（sepharose beads）上仍然有大量的GST-X接合蛋白質，並可以蛋白質切割（factor Xa）將之切割，利用西方點墨法（western blotting）及酵素連結免疫吸附檢驗法（ELISA）檢測分析GST-X接合蛋白質，由GST及X抗體對GST、GST-X及X三種抗原辨識的情形，證實了所合成GST-X接合蛋白質的正確性。為了確認合成的重組蛋白質是否能夠產生具有專一性的抗體，分別以接有GST-X接合蛋白質的醣珠及純化的GST-X接合蛋白質為抗原免疫兔子，兩者皆能夠產生對GST-X接合蛋白質有專一性的抗體，而前者免疫血清的力價（titer）較高。最後，以合成的蛋白質對20位肝癌病人血清進行分析，由西方點墨法檢測結果初步認定，肝癌病人血清中X抗體的力價極低。 Plasmid, pGEX-5x-1-X, containing PCR-amplified hepatitis B virus X antigen gene was constructed in glutathione S-transferase (GST) gene fusion system and expressed in E. coli DH5(. The sequence and orientation of the gene was in frame constructed and confirmed by dideoxy nucleotide sequence. Elevated expression and purification of the recombinant fusion GST-X were performed. After elution, thesepharose beads still contain large amount of the GST-X fusion protein, which was specifically cleaved by restriction protease factor Xa. Immunological characterizations of the fusion protein were studied using anti-GST and anti-X antibodies by Western blotting immunodetection and ELISA. The specific bindings between antibodies and GST,GST-X, and X antigens show the precision of the recombinant fusion protein. Antibodies against the eluted sepharose beads and the purified GST-X protein were raised in rabbits. Both of sera from rabbits contain antibodies specific to the GST-X fusion protein.The former one has a higher titer than the latter one does.In addition, the fusion protein was used to test with the sera from patients with hepatocellular carcinoma (HCC) by Western blotting immunodetection. Results indicate that these sera contain very low titer of antibody with specific binding to the X antigen of the protease-digested GST-fusion protein.
