Construction of recombinant CHO cell strain for high expression of HBsAg / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media.</p><p><b>METHOD</b>Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product.</p><p><b>RESULT</b>CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000.</p><p><b>CONCLUSION</b>Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.</p>

The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus / 病毒学报

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

The investigation of hepatitis D virus infection situation in the human with HBsAg in Foshan of Guangdong province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China.</p><p><b>METHODS</b>ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan.</p><p><b>RESULTS</b>The results from two ELISA kits and RT-PCR were identical. Eight samples were positive.</p><p><b>CONCLUSIONS</b>The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.</p>

Antibody detection of hepatitis E virus in human population of different national in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of HEV infection in different national human population in Han, Hui and Zang in China.</p><p><b>METHODS</b>EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2008 in Sichuan, Beijing, Heilongjianin, Sandong, Gansuo, Ningxia and Qinghai areas.</p><p><b>RESULTS</b>The total positive rate of anti-HEV IgG was 17.97% (1878/ 10448), 24.32% (1794/7376) in Han national, 3.59% (81/2258) of Hui national and 0.37% (3/814) of Zang national, respectively. The positive rate of Han human at different age group, was 5.19% of < or = 10 year, 11.64% of 11-20 year, 20.08% of 21-30 year, 34.17% of 31-40 year, 41.75% of 41-50 year, 48.58% of 51-60 year, 57.43% of > or = 61 year. The positive rate of Hui human at different age group, was 3.11%, 3.96%, 2.11%, 3.98, 2.52%, 4.57% and 6.67%, respectively. Three positive of Zang human was between 21-60 year.</p><p><b>CONCLUSIONS</b>The HEV infection in Han national population was higher than the Hui and Zang national, significantly. The HEV infection was correlation with age significantly, the infection rate was increased with age.</p>

Hepatitis C viruses infection situation in the human population of six provinces in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of hepatitis C viruse infection in the human population in six regions of Beijing, Heilongjiang, Shandong, Ningxia, Gansu and Sichuan in China.</p><p><b>METHODS</b>ELISA was used for detecting anti-HCV IgG of the serum samples. All sample were collected in 2006-2008 in six areas.</p><p><b>RESULTS</b>9538 samples were detected. The total positive rate of anti-HCV was 0.39% (37), 0.23% (3/1328) in Beijing, 0.74% (12/1629) in Heilongjiang, 0.26% (5/1962) in Shandong, 0.1% (1/1000) in Ningxia, 0.44% in Gansu (9/2 037) and Sichuan (7/1582), respectively. The 37 positive samples at the sex were 19 (51.35%) of man and 18 (48.65%) of female. At the age group were 1 (2.70%) of < 10 years old, 5 (13.51%) of 10-19 years old, 4 (10.81%) of 20-29 years old, 6 (16.22% ) of 30-39 years old, 9 (24.32%) of 4049 years old, 12 (32.43%) of > or = 50 years old. The positive samples were detected anti-HAV-IgG, anti-HEV-IgG and HBsAg/ HBcAb /HBeAb. The positive Number was 35 (94.59%), 10 (27.03%) and 2 (5.41%) respectively.</p><p><b>CONCLUSIONS</b>HCV infection rate in the population was 0.1% -0.74%, 1/3 of > or = 50 years old in HCV positive. Hepatitis C virus co-infection with HAV, HEV and HBV.</p>

The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods.</p><p><b>RESULTS</b>The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition.</p><p><b>CONCLUSIONS</b>The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.</p>

Hepatitis viruses infection situation in human population of the Gansu province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of hepatitis viruses in human population of Ganso province.</p><p><b>METHODS</b>ELISA was used for detecting anti-HAY IgG, HBsAg/HBsAb, anti-HCV IgG and anti-HEV IgG of the serum samples. All sample were collected in four areas of KL, LT, HN and ZhL of Gansu province in 2008.</p><p><b>RESULTS</b>1977 samples were detected, The positive rates of anti-HAY, HBsAg/HBsAh, anti-HCV, and anti-HEY are 84.57% (1672/1977), 4.81% (95/1977) and 28.73% (568/1977), 0.46% (9/1977),and 13.10% (259/1977), respectively. The positive rate at different age group, for anti-HAY was 43.21% of c 9 years old, 80.23% of 10-19 years old, 93.02% of 20-29 years old, 95.55% of 30-39 years old, 95.60%-97.52% of 40-60 years old. For HBsAg/HBsAb were 2.09% or 29.27%, 3.02% or 21.81%, 6.20% or 36.43%, 5.93% or 31.16%, 6.43% or 3l.43%, 7.32% or 24.88%, 3.33% or 28.89% at the same age group, respectively, for anti-HCV, was 0.59% of 30-39 years old, 1.79% of 40-49 years old, 0.98% of 50-59 years old. For HEY-IgG was 3.48% of < or =9 years old, 6.05% of 10-19 years old, 8.53% of 20-29 years old, 19.29%-21.67% of 30- > or =60 years old.</p><p><b>CONCLUSIONS</b>The inoculation againt HAY and HBY is enhanced in the youngster population. HBsAg carrier and UCY infection is decreasing. The HEY infection in the Hid people is lower than the Han nationality.</p>

Antibody detection of hepatitis E virus in some human population, swine and chicken in Beijing in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of HEV infection in human population, swine and chicken in Beijing region.</p><p><b>METHODS</b>EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2007 in Beijing areas.</p><p><b>RESULTS</b>The anti-HEV IgG was detected positive in 21.52% of human (260/1208), 46.88 % (15/32) of swine, but was negative in chickens (0/24). The positive rate of human at different age group, was 5.60% (14/250) of 11-20 year, 20% (42/210) of 21-30 year, 24.03% (62/258) of 31-40 year, 26.44% (78/295) of 41-50 year, 32.82% (64/195) of 51-60 year. The male (29.51%) was higher than the female (21.70%).</p><p><b>CONCLUSION</b>The HEV infection was correlation with age and sex significantly. The infection rate was increased with age, the positive rate in swine was more double than the human population.</p>

Antibody detection and sequencing analysis of hepatitis E virus in human population, swine and chicken in Sichuan region in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of HEV infection and genotype.</p><p><b>METHODS</b>ELISA were used for detecting anti-HEV IgG of the serum samples, the nested reverse transcriptase PCR (RT-nPCR) was used for detecting HEV RNA in patient serum and swine bile samples. All samples were collected in 2005-2007 in some districts in Sichuan province. The primers used for genotyping were the ORF2 region of HEV genome.</p><p><b>RESULTS</b>The anti-HEV IgG was detected positive in childrens 6.10% (41/672), adults 42.26% (280/ 661), swines 88.89% (32/36), chickens negative (0/59). 1 case of 15 serum samples of anti-HEV IgM positive and 3 of 54 swine bile samples were positive for HEV RNA by RT-PCR.Sequence analysis of 4 isolates has 92.1% to 98.6% nucleotide sequence homology. These isolates from human and swine were identified closely related to Ch-T21 strain 90.1%-96.9% sequence homology, which belonged to HEV genotype 4B.</p><p><b>CONCLUSIONS</b>The swine were the risk factors in the spread of hepatitis E virus.</p>

Evaluation of the ELISA diagnostic kits for hepatitis E virus antibody in the reference serum, the suspect patients of hepatitis E and normal persons' sera / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the quality of the ELISA diagnostic kits for detecting hepatitis E virus (HEV) specific IgG antibody.</p><p><b>METHODS</b>Five diagnostic kits (WT, GD, HM, GeneLabs and KH) for anti-HEV IgG were assayed by detecting HEV IgG in the HEV diagnostic reference sera from 24 positive cases and 30 negative cases. 42 cases clinical suspect patients with hepatitis E from Ditan hospital in Beijing in 1994 to 1995 and 230 normal persons' sera from Chengdu city, Sichuan province in September in 2005.</p><p><b>RESULTS</b>The conformity rates of positive and negative sera was 95.83% (23) and 96.67% (29) for WT kit, 87.50% (21) and 100% (30) for GD kit, 87.50% (21) and 96.67% (29) for HM kit, 66.67% (16) and 100% (30) for GeneLabs kit, 45.83% (11) and 100% (30) for KH kit for detecting anti-HEV positive and negative reference sera, respectively. The five kits were used to detect anti-HEV IgG among 42 clinical suspect patients with hepatitis E and 230 normal persons' sera, The positive rate was 97.62% (41) and 31.74% (73) by WT, 100% (42) and 17.39% (40) by GD kit, 97.62% (41) and 23.91% (55) by HM kit, 90.48% (38) and 7.83% (18) by GeneLabs kit, 90.48% (38) and 3.48% (8) by KH, respectively.</p><p><b>CONCLUSION</b>The positive rates of all the 5 kits were over 90% and had a very high conformity in detecting anti-HEV IgG in clinical patient with hepatitis E, positive rates of 3.48% to 31.74% were found in detecting the normal persons' sera. The research showed insufficiency of the ELISA kits for epidemiological survey of HEV infection in population.</p>

Expression of human IL-12 in mammalian cell and study on its biological activities / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.</p><p><b>METHODS</b>Bicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites (IRES). pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO-DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA-activated lymphoblasts proliferation assay and IFN-gamma induction assay were used in this study.</p><p><b>RESULTS</b>Genetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efficiency and good growth properties.</p><p><b>CONCLUSION</b>rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.</p>

Expression of the hepatitis Delta antigen in prokaryotic cell and evaluation of its application as an EIA diagnostic reagent / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct the pRSETB-HDAg recombinant expression plasmid and to obtain soluble hepatitis D virus antigen with high biological and antigenic activity.</p><p><b>METHODS</b>HDAg gene fragment was inserted into fusion expression pRSET B vector that includes T7 promoter and a polyhistidine tag. The recombinant plasmid was transformed into host bacterium BL21 after induction with IPTG. The expression supernatant was purified by chelating affinity chromatography and the recombinant HDAg antigenic activity was detected by EIA.</p><p><b>RESULTS</b>EIA detection using the recombinant HDAg showed strong positive reaction with hepatitis D patients sera. The positive rates of the EIA, compared with HDAg from USA and Hua Mei EIA kit in detecting 26 cases of anti-HDV positive reference sera, were 100%, 96.15% and 100%, respectively.</p><p><b>CONCLUSION</b>Recombinant plasmid for HDAg with good antigenicity was successfully constructed and could be used as hepatitis D antibody detection reagent.</p>