Preparation And Application Of Monoclonal Antibodies Against AR Protein

AR is a member of AKR1B group of aldoketo reductases. AR is thought to be involved in diabetic complications and Atherosclerosis. ARL-1 is a NADPH-dependent enzyme like AR, which can deoxidize compounds of aldehyde and dextrose. Relevant researches suggest that AR and ARL-1 possibly relate to liver cancers. To study the relation between AR, ARL-1 and hepatoma, the monoclonal antibodies(mAb) against aldose reductase(AR)protein exclusive aldose reductase-like protein (ARL-1) were prepared.In this study, RNA was extracted from human placenta. The AR gene was gained by RT-PCR and was inserted into the E.coli expression vector pGEX-4T-1 (His) 6C. Recombinant AR proteins named GST-AR were expressed. Because the amino acid sequence of ARL-1 was 71% homogenous to that of AR, to select the mAb against AR better, the parts of AR (80～142 aa), which are different from ARL-1, were expressed by the same methods. And it was designated as GST-dAR. GST-AR and GST-dAR were purified by affinity chromatograph. Simultaneously, to analyze the binding sites of the mAb against AR and AR protein, according to the analysis in antigenicity of AR by software (Clustalx,Antheprot), 4 parts of AR were expressed, respectively named GST-dA1(1～79 aa),GST-dA2(80～99 aa),GST-dA3(111～142 aa),GST-dA4 (143～316 aa). AR protein (GST-AR) was used to immunize BALB/c mouse. And mAb was prepared by hybridoma technique. The anti-AR mAb were detected and applied by ELISA and Western blot comparing with anti-ARL-1 mAb. Finally, five clones secreting specific anti-AR mAbs were obtained, named ARB3, ARE6, AR7B3G4, ARF10 and ARH8 respectively. They were all of IgG1, type ofκ. Western blot showed that all the five mAbs bound to GST-AR protein and had no cross-reaction to GST-ARL-1 and GST protein. ARB3 and ARE6 recognized GST-dA1. AR7B3G4 recognized GST-dA3. ARF10 and ARH8 recognized GST-dA4. AR protein was expressed in placenta, fetal tissues including kidney, brain, lung and liver. And higher expression of AR protein or ARL-1 protein in the liver cancer than matched normal tissues adjacent to tumors was detected.All these results suggest that the mAbs be highly specific anti-AR mAbs recognized the 1~79 Amino Acid sites, 111~142 Amino Acid sites, 143~316 Amino Acid sites of AR respectively. AR or ARL-1 protein was possibly overexpressed in liver cancer. The anti-AR mAb, with the anti-ARL-1 mAb protein together, may be a useful tool for further studying the function of AR, the relationship between AR, ARL-1 and HCC and for further investigation of epidemiology.