ABSTRACT
Objective:To explore the key amino acids of side chain dioxate dehydrogenase complex E2 protein (BCOADC-E2) that can react specifically with specific autoantibody anti-mitochondrial antibodies (AMA)-M2 in patients with primary biliary cholangitis (PBC).Methods:The homologous target gene BCKD, which expressed the epitopes of BCOADC-E2 protein, was cloned and recombined with the engineering plasmid pGEX-4T1. Fifteen point mutant plasmids were obtained by polymerase chain reaction (PCR) and transferred to the prokaryotic expression strain for protein expression and purification. Fourteen mutant proteins and one wild-type protein were obtained. The AMA-M2 specificity of the 14 mutant proteins was measured by enzyme-linked immuno sorbent assay (ELISA), and the amino acids that were critical to the specificity of BCOADC-E2 and AMA-M2 were identified by comparing the specificity of the 14 mutant proteins with that of the wild type proteins. Differences between groups were analyzed by analysis of variance, LSD- t test. Results:A total of 14 mutant proteins and 1 wild-type protein were obtained.The specific reaction degree of mutant protein pGEX-BCKD-S1A (1.634±0.328) and pGEX-BCKD-C3A (1.744±0.345) with serum AMA-M2 in patients with PBC was higher than that of wild-type protein pGEX-BCKD (1.000±0.000) with AMA-M2; Mutant protein pGEX-BCKD-E4A (0.157±0.067), pGEX-BCKD-V5A (0.057±0.029), pGEX-BCKD-Q6A (0.580±0.166), pGEX-BCKD-S7A (0.744 ±0.125), pGEX-BCKD-D8A (0.351 ±0.135), pGEX-BCKD-S10A (0.496 ±0.158), pGEX-BCKD-V11A(0.149±0.089), pGEX-BCKD-T12A(0.061±0.043), pGEX-BCKD-I13A(0.007±0.017), pGEX-BCKD-T14A (0.198±0.101), pGEX-BCKD-S15A (0.156±0.087), The specific reaction degree of pGEX-BCKD-R16A (0.884±0.099) with AMA-M2 was lower than that of wild-type protein pGEX-BCKD (1.000±0.000) with AMA-M2.Conclusion:The cysteines at positions 1 and 3 of BCOADC-E2 protein were the key amino acids to improve the specific reaction between BCOADC-E2 and AMA-M2. The mutant proteins formed after amino acids at position 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15 and 16 are replaced by alanine can decrease the specificity of AMA-M2. Amino acids at positions 5 and 13 are the key amino acids that affect the specific reaction between BCOADC-E2 and AMA-M2, and have an important effect on the function of BCOADC-E2 protein.
ABSTRACT
Objective:To increase the critical relative humidity(CRH) of Qubi granule,thus to improve its humidity resistance. Methods:The best prescription of Qubi granule was optimized by comparative method,and to determine its critical relative humidity. Results:The old prescription of the Qubi granule's CRH was 50%.With new prescription,the Qubi granule’ s CRH was 61%. the latter has increased by 11% as compared with the former . Conclusion:Considering above result,there is a hope to improve the hygroscopicity of Qubi granule.
ABSTRACT
AIM:To study the optimum condition of hydroxypropyl-?-cyclodextrin inclusion for mangiferin in order to improve the solubility of mangiferin. METHODS: By comparing the condition of inclusion of mangiferin in hydroxypropyl-?-cyclodextrin obtained,including its drying method,inclusion compound stood the test of infrared spectrophotometry,UV spectrophotometry,differential scanning calorimetry analysis,phase solubility diagran and solubility determination. RESULTS: The paddling process and freeze drying could put in HP-?CD inclusion compound use the ratio of host/guest molecules was 1∶1,the solubility of mangiferin of included complex was 35.99 mg/mL,about 300 times as large as uninclused mangiferin(0.111 mg/mL). CONCLUSION: Hydroxypropyl-?-cyclodextrin inclused mangiferin compound could be prepared by paddling process and freeze drying.Its solubility of the compound improves significantly and it is stable.