Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB / 中山大学学报(医学科学版)

ObjectiveTo establish a mutation library of rifampicin resistance gene rpoB. MethodsThe ΔrpoB attB：：rpoB strain of Mycobacterium smegmatis （M. smegmatis） be constructed by homologous recombination and L5 attB phage integration site exchange. Based on the L5 attB plasmid exchange system and resistance selection medium， 48 clones are selected to verify plasmid replacement efficiency. Degenerate primers are designed every 3 bases in the rifampicin resistance determining region （RRDR）， and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification. The library fragments are seamlessly cloned into the vector and transformed into Escherichia coli （E. coli）to form an E. coli mutation library. Based on the principle of plasmid exchange， the mutant plasmid library is transformed into the M. smegmatis strain ΔrpoB attB：：rpoB， and the original L5 attB site plasmid is replaced to form the M. smegmatis mutant library. The genotype of the library are determined by genome extraction， library construction and high-throughput sequencing. ResultsCompared with the wild-type rpoB gene （5 600 bp）， the amplified fragment of the rpoB knockout strain is 2 200 bp， which proved that the ΔrpoB attB：：rpoB conditional knockout strain of M. smegmatis is successfully constructed. The success rate of plasmid replacement is 100%. There were 540 kinds of single amino acid mutations in both E. coli library and M. smegmatis library， 5 301 kinds of multi-point mutations in E. coli library， and 853 kinds of multi-point mutations in M. smegmatis library. The correlation coefficient between E. coli library and M. smegmatis library is 0.84. ConclusionsWe have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene rpoB， and successfully established a mutant library of rifampicin resistance gene rpoB.

Variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus during lamivudine treatment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads.</p><p><b>METHODS</b>HBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing.</p><p><b>RESULTS</b>In HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups.</p><p><b>CONCLUSIONS</b>HBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.</p>

Expression, purification and characterization of a phyA(m)-phyCs fusion phytase / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The phyA(m) gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyA(m)-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4+/-0.53) U/ml at the flask scale and (159.1+/-2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 degrees C and an optimal pH at 5.5~6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 degrees C to 95 degrees C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoH(f)), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyA(m).

The associations between polymorphisms of APOBEC3G and different outcomes of persistent HBV infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the associations between polymorphisms of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and different outcomes of HBV infection in the Chinese Han population.</p><p><b>METHODS</b>Six hundred thirty-five chronic hepatitis B patients were divided into 3 groups: 202, 217 and 216 patients were HBV cleared, chronic hepatitis B, and with liver cirrhosis, respectively. Five tagSNPs (rs8177832, rs17000736, rs17496046, rs9622924 and rs2899313) were genotyped by pyrosequencing. HBV viral loads were determined by real-time PCR method. Chi square was used for statistics.</p><p><b>RESULTS</b>The majority of rs8177832 allele was A/A and the frequencies of rs8177832 allele among these groups were not significantly different (P more than 0.05). HBV viral loads were higher in chronic hepatitis B patients with G allele than in chronic hepatitis B patients with A allele (P less than 0.05). The rs17000736 and rs9622924 alleles were found only in G/G and C/C genotypes. There were also no significant differences in the other four SNPs alleles (rs17000736, rs17496046, rs9622924 and rs2899313) in these groups (P more than 0.05).</p><p><b>CONCLUSIONS</b>rs8177832, rs17000736, rs17496046, rs17000736 and rs2899313 of the APOBEC3G gene might not be associated with HBV persistent infection in patients in this study. However, the rs8177832 polymorphism may be involved in inhibiting HBV replication.</p>