Preparation method of hyperoside from Flos Abelmoschus manihot / 国际中医中药杂志

Objective To study the preparation method and analytical technique of hyperoside from Flos Abelmoschus manihot.Methods Hyperoside was isolated and purified by solvent extract and chromatography, whose structure was determined by 1H-NMR and 13C-NMR. The purity was analyzed by TLC and HPLC.Results The TLC showed that the hyperoside had no impurity spot. The HPLC indicated that the purity reached more than 98.5%.Conclusions The mothod of isolation and purification for hyperoside reported in this paper was simple and economical.

Construction of The Antigen Expression Vector Used for Library-library Screening / 生物化学与生物物理进展

Selectively infective phage (SIP) technology was developed for screening interacting protein-protein pairs. The in vivo SIP strategy would in principle be suitable for??library-library??selections, and the co-packaged polyphage may be a suitable approach. In order to construct the antigen expression vector which can be co-packaged into polyphage with phage displaying vectors, plasmid TG10 was chosen as the basic vector which is compatible with antibody display vector. The interval sequence of phage genome was amplified with PCR and cloned into TG10 to provide the packaging signal. It was named pTMI and it can be packaged into phage particles in 1011 level. The N1N2 region of gene ￠? was amplified and cloned into pTMI under the control of lac promoter to give pTMIN. Promoter trc was synthesized and replaced the lac promoter to give pTTMIN which permits the fusion expression of antigen with N1N2. To test its ability for fusion expression, gene code for ten-peptide of c-myc was synthesized and inserted into pTTMIN downstream to N1N2. After induction expression, the results of ELISA and SDS-PAGE showed that it has been expressed successfully. When pTTMIN was transfected into cell carrying antibody display vector p3MHHB3, it was copackaged into phage particles in 0.3% to 55% after rescuing with helper phage VCSM13.From the results it can concluded that the antigen expression vector was constructed successfully and it can be used for library-library screening in theory.

Construction of helper virus VCSM13N1 with amber mutation / 第二军医大学学报

Objective:To construct the helper virus VCSM13N1 with amber mutation,so as to provide a platform for the selective infective phage(SIP) technique.Methods: A pair of primers with amber stop codon were synthesized and introduced into N1 region of helper virus VCSM13 protein by overlapping extension PCR,and the products were identified by ristriction enzyme digestion and sequencing.The switch on/off efficiency of amber codon was detected with E.coli XL1-Blue and HB2151.The feasibility of using the amber mutant phage as helper virus was determined with anti-HBsAg phage antibody.Comprehensive analysis was also made on the performance of the amber mutant phage.Results: The titer of mutant phage was 2.5?10~(11) when detected with E.coli XL1-Blue,comparable with that of wild phage.The titer of mutant phage was at 10~6 level when E.coli HB2151 was used,being 10 000 folds lower than that of wild phage,when the mutant phages from HB2151 were used to infect E.coli XL1-Blue,the titer peaked at 5?10~(9),being 50 folds lower than that of wild phage.The activity of phage antibody prepared with mutant virus was comparable with the phage antibody prepared with wild phage.Conclusion: The constructed amber mutant phage can be used as helper virus,and it can switch off efficiently in non-suppressing strain HB2151.

Construction of a packaged strain XL-pⅢ and preparation of defective helper phage / 解放军医学杂志

Objective To construct a packaged bacteria strain and, with it, to prepare gene III-defective helper phage. Methods Whole gene III and chloramphenicol-resistant gene were amplified and joined to form a fragment flanking 36bp homologous sequence of hisBCD using overlapping extending PCR, and it was then integrated into chromosome of E. coli XL1-Blue to replace the hisBCD gene using Red recombination system. The recombinant strain was identified with chloramphenicol-resistant screening, PCR and histidine-phenotype analysis, and named as XL-pⅢ. The gene III-deleted genome of phage was constructed with PCR and transfected into the recombinant strain XL-pⅢ to prepare the defective helper phage. It was quantified by infecting E. coli XL1-Blue and formed colonies were counted in kanamycin plate. Results The recombinant strain could grow in chloramphenicol-resistant plate, but couldn't grow in M9 medium without histidine, implying that it had lost its histidine phenotype. The aimed gene fragment could be amplified as designed. The constructed recombinant was named as XL-pⅢ. The prepared defective helper phage could infect E. coli XL1-Blue only once and the CFU was 3.7?108. Conclusions The packaged strain XL-pⅢ is successfully constructed and used to prepare the gene III-deleted helper phage. It is expected to be used in SIP system.