Effect of Yunnan herb Laggera pterodonta against influenza A (H1N1) virus in vitro / 中国中药杂志

Laggera pterodonta is commonly used for treating influenza in Southwest China, especially in Yunnnan province. The main clinical effects of L. pterodonta include anti-influenza, anti-microbial, anti-inflammatory. To investigate the anti-influenza A (H1N1) virus effect of L. pterodonta, neutralization inhibition and proliferation inhibition tests were performed. MDCK culture method was used to observe the cytopathic effect (CPE) of extracts from L. pterodonta in inhibiting influenza A (H1N1) virus and haemagglutination titre of H1N1 virus in vitro. The culture medium were collected at 24 h, 48 h, 72 h, 96 h, and detected by Real time RT-PCR, in order to compare the effect of different extracts from L. pterodonta on in vitro proliferation of H1N1, virus. The result of neutralization inhibition test showed that hemagglutination titer of ethyl acetate extract were 8 times lower at 72 h; in proliferation inhibition test, hemagglutination titer of ethyl acetate extracts reduced by 2 and 4 times. According to the results of Real time RT-PCR test, the H1N1 inhibition ratio of ethyl acetate extract was 72.5%, while the proliferation inhibition ratio of ethyl acetate extract was 25.3%; as for petroleum ether extracts, the H1N1 inhibition ratio was 60.2%, while the proliferation inhibition ratio was 81.4%. In conclusion, both ethyl acetate extract and petroleum ether extract of L. pterodonta have significant neutralization and direct proliferation inhibition effects on influenza A virus.

Complete genome sequence characteristics of human echovirus 9 strain isolated in Yunnan, China / 病毒学报

To analyze the genomic sequence characteristics of a human Echovirus 9(ECHO-9) strain isolated from a child with Hand-foot-mouth disease (HFMD) in Kunming, Yunnan Province, in 2010. The complete genome sequence of a human echovirus 9 strain, MSH-KM812-2010 was determined. As other human enterovirus, its genome was 7,424 nucleotides (nts) in length and encoded for 2,203 amino acids (aas). In comparison to other human enteroviruses, MSH-KM812-2010 strain had the highest homology with other strains of human echovirus 9 in structural genomic regions and more homologous to other serotypes of B specie than to human echovirus 9 in non-structural genomic regions. Phylogenetic analysis based on complete VP1 gene revealed that the sequences of human echovirus 9 segregated into three distinct clades A, B and C with more than 15. 0% diversity between clades. All Chinese isolates belonged to the same clade. RDP3 and Blast revealed evident recombination in non-structural genomic regions. This report is the first to, describe the complete genome of the human echovirus 9 in China and provide an overview of the diversity of genetic characteristics of a circulating human echovirus 9.

Adaptability and purification of dengue-III virus D9964 strain in KMB17 cells and proliferation kinetics of adapted strain / 病毒学报

To select the adaptive strain of Dengue-III virus D9964 strain (China strain) in KMB17 cells, elucidate the biological characteristics and proliferation kinetics of adapted strain,and to lay the foundation for the development dengue inactivated vaccine and attenuated live vaccine. Dengue-III virus D9964 strain was firstly identified by amplification of the type-specific gene segment of dengue virus by RT-PCR, and the titer was determined. The virus was then subcultured in KMB17 cells with 4.0 MOI till completely adaptive to multiply in cell S. After subculturing in KMB17 cells for 10 consecutive passages, the adapted strain was screened, and purified through plaque. Virus titer of each passage was measured by microtitrimetry, and the antigenicity was detected by IFA. The purified virus RNA extraction of 3-8 day cultured from KMB17 cells, was performed to detect the proliferation kinetics of adapted strain. The results showed that after continuous subculture, dengue-III virus D9964 (China) strain could stably proliferate in KMB17 cells, a highly puried virus adapted strain was obtained through plaque purification. Purified strain maintained the good antigenicity with a highest replicating activity during the 5th-6th day.

Complete nucleotide sequence of a human coxsackievirus A16 strain KMM08 isolated in Kunming, China in 2008 / 中华流行病学杂志

Objective To describe the genetic characterization of complete genome from a human coxsackievirus A16 (CA16) strain KMM08,isolated in Yunnan,China,in 2008.Methods By using RT-PCR,the seven fragments contained about 1000 nucleotides in the complete genome were sequenced.The sequences were aligned with other enterovirus sequences downloaded from GenBank using Mega 4.1,RDP3 and SimPlot 3.5.1 software.Results As in other human enterovirus,its genome was 7409 nucleotides in length,encoding for 2193 amino acids.KMM08 strain was closely related to other reference strains of B genotype.In the complete genome,the homology of nucleotide and amino acid among the eleven CA16 isolated strains were 79.0％-98.2％ and 94.5％-99.3％,respectively.The rates of homology were 79.1％ and 94.8％ when comparing with that of G10 strains and 78.7％ and 89.0％ comparing with that of BrCr strains,respectively.SZ-HK08-3 strain had high homology when compared to other strains.In different segment of genome,the rates of homology were 97.0％-99.0％ and 98.0％-100.0％ when compared with that of SZ-HK08-3 strains,respectively.The rates of homology were 74.2％-86.9％ and 90.9％-97.0％ when compared with that of G10 strains,respectively and were 65.0％-84.9％ and 71.0％-95.2％ when compared with that of BrCr strains.Data from Phylogenetic analysis showed that KMM08 belong to genotype B.The putative recombinant Tainan-5079-98 was detected positive with RDP3 and SimPlot 3.5.1.Conclusion KMM08 strains isolated in Yunnan in 2008 belonged to B genotype of coxsackivirus A16.The possible occurrence of inter-typic recombination would involve EV71 and CA16.

The analysis of human papillomavirus type 16 E6/E7 genetic variability in Yunnan Province, China / 病毒学报

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.

RNA interference-mediated inhibition of survivin expression in Hela cell line by siRNA expression vector targeting survivin gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare small interfering RNA (siRNA) targeting survivin for inhibition of endogenous survivin gene expression in Hela cell line and evaluate its effect on promoting Hela cell apoptosis.</p><p><b>METHODS</b>The recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into Hela cells, in which the expression level of survivin was determined by immunofluorescence staining and survivin gene transcription detected by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>Introduction of the plasmids pshRNA-survivin-1 and pshRNA-survivin-2 into Hela cells resulted in efficient and specific inhibition of survivin expression as demonstrated by immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced. In contrast, the control plasmid did not exhibit any inhibitory effect on the protein expression and mRNA transcription of survivin gene. PI-Annexin V staining indicated an apoptosis rate of the transfected Hela cells of (36.02-/+2.12)% (P<0.01) and (35.29-/+2.02)% (P<0.01), respectively.</p><p><b>CONCLUSION</b>The prepared siRNA targeting survivin gene is capable of inducing marked inhibitions of survivin protein expression and RNA transcription and significant enhancement of apoptosis in Hela cells, which shed light on a new strategy in gene silence therapy targeting survivin.</p>

Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).</p><p><b>METHODS</b>The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.</p><p><b>RESULTS</b>The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.</p><p><b>CONCLUSION</b>The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.</p>

Refolding and purification of the huGM-CSF(9-127)-IL-6(29-184) fusion protein / 生物工程学报

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.