Analysis of classical prescription Jinshui Liujun Jian based on ancient literature / 中国中药杂志

To provide the ancient literary evidence support for the clinical application and development of classical prescription based on systematical collection and analysis of the ancient Chinese medical literature containing Jinshui Liujun Jian, including its origin and development. Bibliometric analysis was used and information of Jinshui Liujun Jian in ancient Chinese medical literature was then collected for statistical analysis of formula compositions, main indications, dosage, preparation methods, etc. A total of 151 valid items of data were obtained from 48 ancient Chinese medicine books. Jinshui Liujun Jian was first recorded in Jingyue Quanshu written by ZHANG Jiebin. This prescription consisted of Rehmanniae Radix Praeparata, Angelicae Sinensis Radix, Pinelliae Rhizome, Citri Reticulatae Pericarpium, Poria and Glycyrrhizae Radix et Rhizome Praeparata cum Melle, and it was mainly used to treat the deficiency of lung and kidney, edema and excess production of phlegm, or Yin deficiency in the old, insufficient blood-qi, wind-cold evil, cough and disgusting, asthma and excessive phlegm. Doctors in later dynasties mostly followed the prescription compositions, dosages and indications in Jingyue Quanshu, and extended the clinical application of this prescription.

Antivirus effect of plasmid co expressing hepatitis B surface antigen and granulocyte macrophage-colony stimulating factor / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether GM-CSF can enhance the antiviral effect of HBsAg DNA vaccine.</p><p><b>METHODS</b>Divided animals into 8 groups. Group A: pcDNA3.1-S 100microg; Group B: pcDNA3.1-GM-CSF-S 100microg; Group C: pcDNA3.1-S-GM-CSF 100microg; Group D: pcDNA3.1-S 50microg + pcDNA3.1-GM-CSF 50microg; Group E: pcDNA3.1-GM-CSF 100microg; Group F: recombinant HBsAg vaccine 1microg; Group G: pcDNA3.1,100microg; Group H: PBS 100microl. Serum HBsAg level and concentration of IL-2, IL-4 and IFN-gamma were examined using commercial ELISA kit. The [3H] thymidine incorporation into DNA of Spleen cells was measured; HBsAg expression of hepatocytes from HBV-transgenic mice was assessed using immunohistochemical analysis.</p><p><b>RESULTS</b>Serum HBsAg level was lower and concentration of IL-2, IFN-gamma and SI was higher in mice immunized with pcDNA3.1-GM-CSF-S than those from mice immunized with pcDNA3.1-S and other groups (F=11.262, P<0.01, F=8.147, P<0.01, F=62.275, P<0.01, F=116.28, P<0.01. Less Hepatic HBsAg expression and decline of pcDNA3.1-GM-CSF-S of mice immunized with pcDNA3 were observed in comparison with control groups (F=41.439, P<0.01). Liver histological analysis showed no evidence of necrosis or inflammation in various groups.</p><p><b>CONCLUSION</b>The plasmid co expressing GM-CSF and HBsAg could improve HBsAg-specific humoral and cellular immune responses induced by plasmid encoding HBsAg alone and enhance HBsAg DNA vaccine antivirus effect.</p>

PreS/S gene mutations in HBV in children infected through mother-to-infant transmission and in their mothers / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the characteristics of mutations in PreS/S gene of HBV in children infected through mother-to-infant transmission and in their mothers with different degree of viremia.</p><p><b>METHODS</b>There were 15 pairs of child and mother in this study. Mothers of all children were chronic asymptomatic HBsAg carrier (ASC) before pregnancy and the children were not inoculated against HBV after birth. Anti-HBV medicine was never administrated to all subjects. The serological markers of hepatitis A, B, C, D and E virus were tested and the titers of serum HBV DNA were quantitated. PreS/S gene was amplified by PCR and cloned into pGEM-T vector with T-A cloning technique. The recombinant plasmid pGEM-PreS/S was confirmed by digestion with restriction enzyme ApaI and SacI. Two clones were selected to be sequenced from each patient.</p><p><b>RESULTS</b>According to the degree of viremia in every pair of mother and child, 15 pairs of child and mother were divided into three groups: group A (both children and mothers had high viremia with HBeAg-positive), group B (high in children and low in mothers with anti-HBe positive), and group C (low in children and high in mothers), and there were 5 pairs in each group. The subtype of each pair was the same. There were 4/5 pairs of HBV with B/adw2 and 1/5 pair of HBV with C/adrq+ in each group. It was shown that there were no difference among the four high viremia groups or between the two low viremia groups in the number of mutations and the number of mutational positions. However, there was significant difference between high viremia group and low viremia group. The mutation was not related to age. There were 56 mutational positions and there was no mutational hotspot in high viremia patients. In two low viremia groups (the mothers in group B and the children in group C), there were 113 mutational positions and 85 mutational positions were hotspots (owned by 5/8 clones in each) which could make 37 amino acids changed. Most of mutational amino acids were located within T and B cell epitopes of envelope protein or/and located in the surrounding regions.</p><p><b>CONCLUSIONS</b>There are many differences in HBV with different degree of viremia, even if it comes from the same strain. There are some regular patterns in the mutations of HBV after HBeAg seroconversion happened.</p>

Specific immune responses in Balb/c mice induced by plasmid coexpressing hepatitis B surface antigen and granulocyte-macrophage colony stimulating factor / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the improvement of specific immune responses induced by plasmid coexpressing hepatitis B surface antigen (HBsAg) and granulocyte-macrophage colony stimulating factor (GM-CSF).</p><p><b>METHODS</b>All Balb/c (H-2d) mice were immunized with pGM-CSF/S, pS/GM-CSF, pS or control plasmids. 4 weeks later, anti-HBs titer and the levels of IL-2, IL-4 and IFN-gamma in the supernatant of splenocytes were detected using enzyme- linked immunosorbent assay (ELISA), and HBsAg-specific cytotoxic T lymphocytes (CTL) activity was measured with a 51Cr release assay, using P815/S transfectants as target cells.</p><p><b>RESULTS</b>The anti-HBs antibody titers in the serum, the levels of IL-2 and IFN-gamma, and the CTL activity in pcDNA3.1-GM-CSF-S immuned mice were higher than those in PcDNA3.1-S immunized mice (F=4.176, P<0.01; F=31.188, P<0.01; F=31.796, P<0.01; F<or=26.891, P<0.01).</p><p><b>CONCLUSION</b>It will improve the specific immune responses induced by HBsAg DNA vaccine after it is binded to the gene of GM-CSF.</p>

Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein / 生物工程学报

To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K-hsp70 was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The result protein was secreted into the supernatant induced by 0.5% methanol at 30 degrees C and purified by centrifugation, ultrafiltration and ATP-agarose. The recombinant Hsp70 was identified by SDS-PAGE, Western blot, mice experiment and effect on the immature DC. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed Hsp70 was about 70 kD and the expressed protein could specifically react with anti-Mt. Hsp70 IgG. And mice immunization indicated the expressed hsp70 had immunogenicity. Hsp70 could induce DC maturation and release Th1 cytokine. The secreted 70 kD protein was about 120 mg/L which accounted for more than 30% of the total supernatant protein and was purified to electrophoretic purity. The Hsp70, which had the biological activity, is successfully secretorily expressed in the Pichia pastoris GS115.

Prokaryocytic expression of the shortened hepatitis B surface antigen and antigenic analysis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the expression of the shortened hepatitis B surface antigen in prokaryocyte and detect the antigenic characters.</p><p><b>METHODS</b>Firstly, the gene fragments coding the 152 and 124 amino acids of the carboxyl terminus of hepatitis B surface antigen (HBsAg) were amplified by polymerase chain reaction (PCR). Secondly, they were cloned to plasmid pBKS+, and the accuracy of those constructions were confirmed by restriction enzyme digestion and DNA sequencing. Then, they were cloned to prokaryocytic expression vector-plasmid pET32a(+). The recombinant plasmids were transfected into E.coli BL21 and induced to express with IPTG.</p><p><b>RESULTS</b>The recombinant plasmids were successfully constructed. In E.coli BL21, the protein was expressed in a fusion fashion and could be recognized by monoclonal antibody against HBsAg with ELISA and Western blot.</p><p><b>CONCLUSION</b>The shortened HBsAg can be expressed in prokaryocyte.</p>

Establishment of consensus sequence of PreS/S of hepatitis B virus with genotype B/serotype adw2 or genotype C/serotype adrq+ prevailing in Chongqing of China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To develop consensus sequence of HBV PreS/S with different subtype in Chongqing of China.</p><p><b>METHODS</b>The gene of PreS/S of HBV in 18 AsC was sequenced. The genotype and serotype of HBV were determined. The main HBV strain prevailing in Chongqing was identified to establish its consensus sequence.</p><p><b>RESULTS</b>It was found that 9 strains were genotype B/serotype adw2, 6 were genotype C/serotype adrq+ and 3 were genotype B/serotype ayw1. The consensus sequence of PreS/S of HBV with genotype B/serotype adw2 and genotype C/serotype adrq+ were established. There were 6 nucleotide variants which causing 4 amino acids change between consensus sequence of PreS/S with genotype B/serotype adw2 in Chongqing and in Southeast of China (homology showed 99.5% and 99.0% respectively). There were 13 different sites that caused 4 amino acids change between consensus sequence of PreS/S with genotype C/serotype adrq+ in Chongqing and in Northeast and South of China (homology showed 98.9% and 99.0% respectively). Comparing the two consensus sequence of PreS/S gene of different subtype HBV in Chongqing, there was 95 variants which caused 40 amino acids variants (the homology was 92.1% and 90.0% respectively).</p><p><b>CONCLUSION</b>The consensus sequence of PreS/S of hepatitis B virus with genotype B/serotype adw2 and genotype C/serotype adrq+ prevailing in Chongqing was established.</p>