[The Application of Estrogen Receptor Aptamer in Immunohistochemical Detection of Breast Cancer].

OBJECTIVE: To synthesize and select an estrogen receptors aptamer that can be used in immunostaining of breast cancer tissues. METHODS: ER protein was purified. ER aptamer that showed a high affinity and specificity for ER was synthesized and selected and by SELEX. Ligand -receptor interactions assay was adopted to measure the affinity of the aptamer-ER complex. Both the biotinylated aptamer and the anti-ER monoclonal antibody were tested for immunohistochemical staining of ER status on 105 breast cancer samples. Agreement on the detection of ER expression was determined by Kappa statistics. RESULTS: The dissociation contant (Kd) of the biotinylated aptamer-ER complex, as calculated by a linear regression analysis, was determined to be (0.34±0.05) nmol/L ( n=3, r=0.989). The binding capacity (B max) was 769.23 fmol/(mg prot·nmol -1·L -1). The ER aptamer and the anti-ER antibody both exhibited identical specificity to ER-expressing breast cancer cells. There was a high agreement between the two methods ( n=105, Kappa value=0.943, 95% confident interval=0.879-1.006, P<0.05 for the ER positive and negtive samples; n=75, Kappa value=0.805, 95% confident interval=0.642-0.967, P<0.05 for the ER weak and moderate/strong expression samples). Both the anti-ER antibody and the ER aptamer can also recognized breast cancer cells at the same sites. There was no expression in the negative controls. There were also positive expressions in the 2 endometrial cancer tissues by using biotinylated aptamer. CONCLUSIONS: Our results indicated that the synthesized ER aptamer has a high affinity to bind ER. ER aptamer and the anti-ER antibody can both be used for immunohistochemical staining of ER status in breast cancer tissue.

Expression of TRAIL in liver tissue from patients with different outcomes of HBV infection.

OBJECTIVES: Hepatitis B virus (HBV) infection triggers the production of TRAIL, suggesting that TRAIL may play a role in liver injury after HBV infection. However, it remains unclear whether TRAIL expression in liver tissue correlates with the extent of liver injury caused by HBV infection. The aim of this article was to investigate the correlation of TRAIL expression and disease severity. METHODS: Liver biopsy specimens were collected from 71 patients with different outcomes of HBV infection, including 25 cases of chronic hepatitis B (CHB), 18 cases of severe hepatitis B (SHB), and 28 cases of liver cirrhosis (LC). Besides, specimens from 33 healthy individuals without detectable liver diseases were used as negative control (NC). The expression of TRAIL was measured by immunohistochemistry. RESULTS: Expression of TRAIL in the HBV-infected patients was higher than that in the NC (P<0.001). Among the patients, TRAIL expression in the ones with CHB was significantly higher than that in NC (P<0.001). However, there was no statistically significant difference between patients with SHB and NC or between the ones with LC and NC (P=0.067 and P=0.178, respectively). Moreover, TRAIL expression in patients with CHB was higher than that in patients with SHB or LC (P<0.001 for both), whereas no statistically significant difference was observed between patients with SHB and the ones with LC (P=0.511). CONCLUSION: TRAIL is involved in the inflammatory and immunoregulatory response after HBV infection. However, there was no significant correlation between expression of TRAIL and the extent of liver injury.

Clinical significance of 4 patients with chronic hepatitis B achieving HBsAg clearance by treated with pegylated interferon alpha-2a for less than 1 year: a short report.

We report 4 chinese patients with hepatitis B e antigen-positive chronic hepatitis B achieving clearance of HBsAg by using pegylated interferon alpha-2a for less than 1 year, to provide one clinical clue for the treatment of chronic hepatitis B.

Elective caesarean section versus vaginal delivery for preventing mother to child transmission of hepatitis B virus--a systematic review.

BACKGROUND: Caesarean section before labor or before ruptured membranes ("elective caesarean section", or ECS) has been introduced as an intervention for preventing mother-to-child transmission (MTCT) of hepatitis B virus (HBV). Currently, no evidence that ECS versus vaginal delivery reduces the rate of MTCT of HBV has been generally provided. The aim of this review is to assess, from randomized control trails (RCTs), the efficacy and safety of ECS versus vaginal delivery in preventing mother-to-child HBV transmission. RESULTS: We searched Cochrane Pregnancy and Childbirth Group's Trials Register (January, 2008), the Cochrane Central Register of Controlled Trials (the Cochrane Library 2008, issue 1), PubMed (1950 to 2008), EMBASE (1974 to 2008), Chinese Biomedical Literature Database (CBM) (1975 to 2008), China National Knowledge Infrastructure (CNKI) (1979 to 2008), VIP database (1989 to 2008), as well as reference lists of relevant studies. Finally, four randomized trails involving 789 people were included. Based on meta-analysis, There was strong evidence that ECS versus vaginal delivery could effectively reduce the rate of MTCT of HBV (ECS: 10.5%; vaginal delivery: 28.0%). The difference between the two groups (ECS versus vaginal delivery) had statistical significance (RR 0.41, 95% CI 0.28 to 0.60, P < 0.000001). No data regarding maternal morbidity or infant morbidity according to mode of delivery were available. CONCLUSION: ECS appears to be effective in preventing MTCT of HBV and no postpartum morbidity (PPM) was reported. However, the conclusions of this review must be considered with great caution due to high risk of bias in each included study (graded C).

[The study about immune response to Balb/c mice inoculated with hepatitis B virus DNA vaccine pCI/S2S].

OBJECTIVE: To construct the hepatitis B virus (HBV) DNA vaccine pCI/S2S, and study the expression of specific antigen protein in muscle tissue section and the specific immune responses of humoral and cellular immunity in BALB/c mice being inoculated with pCI/S2S. METHODS: DNA templates were acquired from the mixed serum of patients with chronic hepatitis B, and the gene fragment S2S of HBV was cloned. The pCI vector and DNA S2S fragment were doubly cut by Kpn I and Not I, and then linked. HBV DNA vaccine pCI/S2S were intramuscularly injected into BALB/c mice, and boosted after 4 and 8 week. The expressing protein product in muscle tissue in situ and the specific cellular immune response were detected in 12 week after the first injection, the specific humoral immune responses were detected at 1, 2, 4, 6, 8, 12 week after the first injection. RESULTS: After pCI/S2S intramuscularly injected into BALB/c mice, HBsAb and preS2Ab were gradually determined with cytotoxic T lymphocyte responses induced, and HBsAg protein was expressed in situ in muscle tissue. CONCLUSION: pCI/S2S is effective HBV DNA vaccine that can induce the specific humoral and cellular immune responses.

[Immunoprotection role of a hepatitis B virus DNA vaccine responsible to BAlb/c mice].

OBJECTIVE: To investigate the specific immune response and the immunoprotection effects of cytotoxic T-lymphocyte (CTL), which is induced by hepatitis B virus (HBV) DNA vaccine pCMV-S2S, on BALB/c mouse with the attack of SP2/0-S2S cells, a BALB/c mouse myeloma cell line stably expressing the HBV preS2S antigen. METHODS: BALB/c mice were divided into 3 groups: experimental group (intramuscularly injected with pCMV-S2S), control group 1 (HBsAg vaccine immunizing) or control group 2 (plasmid pCMV immunizing), which were immunized thrice at week 0.4 and 8 respectively. Specific CTL activities of 3 mice of each group were measured by lactate dehydrogenase (LDH) release assays, 4w after the last boost. The other mice of each group were subcutaneously inoculated with SP2/0-S2S cells into one lateral of flanks. SP2/0-CMV cell without expressing preS2S was subcutaneously inoculated into another lateral of mouse flanks. The time of visible tumor formation and the size of tumor were recorded and observed. The life span and survival rate of mice after loaded with the tumor cells were analyzed by Log-Rank statistics and Kaplan-Meier Survival curve respectively. RESULTS: The specific CTL lysis value of pCMV-S2S group mice was (34.21 +/- 1.38)%, higher than that of HBsAg group [(19.64 +/- 1.50)%] or pCMV group [(3.45 +/- 1.89)%] (P < 0.05). The visible SP2/0-S2S tumor formation rate in pCMV-S2S group mice was 58.3% lower than that in the pCMV group (91.7%), 10 d after inoculated with the tumor cells (P < 0.05). The tumor formation rates of the two control groups were no obvious difference. The date of the first mouse dying in pCMV-S2S group was 24d, delayed for 13d, compared with that in the two control groups. The average life span of pCMV-S2S group mice after loaded with the tumor cells was (31 +/- 1) d, longer than that of the two control groups (P < 0.05). The 4w survival rate of the pCMV-S2S group mice was obviously increased (75%), compared with the two control groups (P < 0.05). The average life span and the 4w survival rate of the two control group mice were no obvious difference. CONCLUSION: The hepatitis B virus DNA pCMV-S2S vaccination may elicit substantial HBV preS2S-specific CTL response in vivo, which gives the mouse the immunoprotection against the attack of SP2/0-S2S cells. The results indicate that pCMV-S2S may be used to the immunoprotection from HBV infection.

[Experimental study of liver fibrosis reversal effect of warming-yang compound formula ganzhifu].

OBJECTIVE: To observe the effect of Ganzhifu (GZF), a Chinese compound formula for Warming-yang on experimental hepatic fibrosis, and to explore its mechanism. METHODS: Fifty-nine Wistar rats were divided into the normal control group and the model group. After the hepatic fibrosis model was established in rats by subcutaneous injection of CCl4 and feeding 10% alcohol in the model group, the rats were randomly sub-divided into the Ganzhifu group, colchicine group and model group, they were treated with Ganzhifu, colchicine and normal saline of same volume once a day respectively for 30 days as one treatment course. The serum liver function and hepatic fibrosis indexes, liver tissue contents of hydroxyproline (Hyp), malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured at the end of the experiment. Histologic changes of liver tissue were observed and scored by the modified inflammatory activity and fibrosis semiquantative scoring system (SSS), and collagen type I , III and IV in liver tissue were stained and semiquantitive analysis conducted. RESULTS: In the Ganzhifu group, liver function and inflammatory activity and fibrosis SSS scores were markedly improved, indexes of hepatic fibrosis, liver contents of Hyp, MDA and collagen type I, III and IV were significantly decreased, and the SOD activity was increased, the changes were more significant as compared with those in the model group. The protective effect of Ganzhifu against hepatic fibrosis was the same as that of colchicine. CONCLUSION: Ganzhifu can alleviate or reverse hepatic fibrosis in experimental hepatic fibrosis, and antioxidation may be its mechanism.

[On the role of liver-enriched transcription factors in regulating HBV transcription and replication].

OBJECTIVE: To investigate the effects of various liver-enriched transcription factors in regulating HBV transcription and replication, and to explore their potential roles in HBV hepatotropism. METHODS: The replication-competent HBV recombinant plasmid pHBV4.1 plus different liver-enriched transcription factor (HNF1, HNF3, HNF4, HNF6, C/EBP and RXRa/PPARa) expression plasmids were co-transfected into nonhepatic cell lines (NIH3T3, HeLa, 293T, SW1353, CV-1 and COS1). The transcription levels of 3.5 kb, 2.4/2.1 kb and 0.7 kb HBV RNA were analyzed by Northern blot hybridization, and the level of HBV DNA replication intermediates was detected by Southern blot hybridization analysis. RESULTS: In the absence of co-transfected liver enriched transcription factor expression vectors, the 3.5 kb HBV RNA is not transcribed and HBV DNA replication is not detected after transfecting of NIH 3T3 cells with pHBV4.1. Expression of the liver-enriched transcription factor HNF4 or RXRalpha/PPARalpha, stimulates the transcription of 3.5 kb HBV RNA and the replication of HBV DNA. In contrast, expression of HNF1, HNF3, HNF6 and C/EBP does not stimulate the transcription of 3.5 kb HBV RNA and therefore does not activate viral replication. HNF4 and RXRalpha/PPARalpha were also shown to activate the transcription of 3.5 kb HBV RNA and viral replication in divers cell types including HeLa, 293T, SW1353, CV-1 and COS1 cells. Mutation of the proximal nucleocapsid HNF4 binding site results in a greatly decreased level of HNF4 or RXRalpha/PPARalpha dependent HBV replication. CONCLUSION: This study demonstrated that the liver-enriched transcription factors HNF4 and RXRa/PPARa can support HBV transcription and replication in nonhepatic cells, indicating that liver-specific gene transcription is one of the determinants of HBV hepatotropism.

[Immune response enhanced by genes encoding IFN-alpha 8 and T alpha 1 co-inoculated with HBV DNA vaccine].

OBJECTIVES: To evaluate if the humoral immune response of hepatitis B DNA vaccine pVAX1-S2S could be enhanced by Talpha1 and/or IFNa expression plasmid co-inoculated. METHODS: The following mammalian expression recombinant plasmids were constructed: the plasmid pVAX1-S2S expressing hepatitis B surface antigen S2S, the plasmid pVAX1-T/I co-expressing thymosin a and IFNalpha, the plasmid pVAX1-I/S2S co-expressing IFNalpha and S2S. These plasmids were inoculated intramuscularly into several BALB/c mice groups in different combinations. In the co-immunization group 1 (pVAX1-I/S2S), each mouse was inoculated with 100 microg of pVAX1-I/S2S; in the co-immunization group 2 (pVAX1-S2S) each mouse was co-inoculated with pVAX1-S2S and 50 microg of pVAX1-TI; in the control group each mouse was inoculated with 100 microg of pVAX1-S2S. All the immunizations were boosted at 2 and 4 week intervals; then the serum samples were collected to detect the anti-HBs and anti-preS2 strengths. RESULTS: 3, 5 and 8 weeks after the first inoculation, the positive rates of anti-HBs were 12.5%, 12.5%, 62.5% respectively in the co-immunization group 1 and 25%, 50%, 50% in the co-immunization group 2, while those in the control group were 0, 25%, 37.5%. The titers of anti-preS2 in co-immunization group 2 was 5 times higher than those in the other two groups. CONCLUSION: The data shows that Talpha1 and/or IFNalpha expression plasmid co-inoculated with pVAX1-S2S might act as an adjuvant to enhance the humoral immune response induced by pVAX1-S2S.

[Influence of wen-yang herbs on hemodynamics in liver fibrotic rats].

OBJECTIVES: To observe the influence of wen-yang herbs on the hemodynamics in liver fibrotic rats. METHODS: Wistar rats with liver fibrosis, induced by carbon tetrachloride and alcohol, were randomly divided into a treatment group and a control group. The treatment group was administered wen-yang herbs and the control group saline. At the end of the experiment, the hemodynamic markers of the liver and the mesentery, the liver function and hydroxyproline content of liver tissues between the two groups were compared. Blood volume of the livers and hydroxyproline content of liver tissues were also determined. RESULTS: Blood volume of the liver and mesentery (P < 0.01) and blood flow velocity of small vein of mesentery (P < 0.05) of the treatment group were distinctly higher than the control group. The hydroxyproline content (P < 0.01) of the treatment group was remarkably reduced and liver function was improved. CONCLUSION: Wen-yang herbs can activate microcirculation of the liver and mesentery, decrease the deposit of collagen in the liver and improve liver function.

Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma.

AIM: To study the expression profiles of HBsAg, HBcAg, p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocar-cinogenesis. METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBcAg, p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, para-cancerous tissues and adjacent normal liver tissues of 40 HCCs were comparatively examined. RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (chi2 =12.774, P < 0.01; chi2 = 18.442, P < 0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues (chi2 = 9.482, P < 0.01; chi2 = 14.645, P < 0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%, which was higher than that in para-cancerous and adjacent normal liver tissues (chi2 = 7.439, P < 0.01; chi2 = 11.174, P < 0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (chi2 = 0.072, P < 0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was 42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (chi2 = 10.551, P < 0.01; chi2 = 18.353, P < 0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (chi2 = 0.014, P > 0.05; chi2 = 0.017, P > 0.05). CONCLUSION: Expression deletion of HBsAg, HBcAg, p21 and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.

Early diagnosis of bacterial and fungal infection in chronic cholestatic hepatitis B.

AIM: To investigate the early diagnostic methods of bacterial and fungal infection in patients with chronic cholestatic hepatitis B. METHODS: One hundred and one adult in-patients with chronic hepatitis B were studied and divided into 3 groups: direct bilirubin (DBil)/total bilirubin (TBil) > or = 0.5, without bacterial and fungal infection (group A, n=38); DBil/TBil <0.5, without bacterial and fungal infection (group B, n=23); DBil/TBil> or = 0.5, with bacterial or fungal infection (group C, n=40). The serum biochemical index and pulse rate were analyzed. RESULTS: Level of TBil, DBil, alkaline phosphatase (ALP) and DBil/ALP in group A increased compared with that in group B. The level of ALP in group C decreased compared with that in group A, whereas the level of TBil, DBil and DBil/ALP increased (ALP: 156+/-43, 199+/-68, respectively, P<0.05; TBil: 370+/-227, 220+/-206, respectively, P<0.01; DBil: 214+/-143, 146+/-136, respectively, P<0.01; DBil/ALP: 1.65+/-1.05, 0.78+/-0.70, respectively, P<0.001). The level of DBil and infection affected DBil/ALP. Independent of the effect of DBil, infection caused DBil/ALP to rise (P<0.05). The pulse rate in group A decreased compared with that in group B (63.7+/-6.4, 77.7+/-11.4, respectively, P<0.001), and the pulse rate in group C increased compared with that in group A (81.2+/-12.2, 63.7+/-6.4, respectively, P<0.001). The equation (infection=0.218 pusle rate +1.064 DBil/ALP -16.361), with total accuracy of 85.5%, was obtained from stepwise logistic regression. Pulse rate (> or =80/min) and DBil/ALP (> or =1.0) were used to screen infection. The sensitivity was 62.5% and 64.7% respectively, and the specificity was 100% and 82.8% respectively. CONCLUSION: Bacterial and fungal infection deteriorate jaundice and increase pulse rate, decrease serum ALP and increase DBil/ALP. Pulse rate, DBil/ALP and the equation (infection=0.218 pusle rate+1.064 DBil/ALP-16.361) are helpful to early diagnosis of bacterial and fungal infection in patients with chronic cholestatic hepatitis B.

[Study of infusion of oxygen-enriched liquid to correct severe hypoxemia in infectious diseases: a report of pilot clinical study].

OBJECTIVE: To investigate a new therapy for effectively correcting severe hypoxemia in patients with infectious diseases by infusion of oxygen-enriched liquid, in order to raise the partial pressure of blood oxygen without passing through pathologically damaged alveoli of such patients. METHODS: Intravenous drip with oxygen-enriched liquids was given to 6 cases suffering from severe acute respiratory syndrome (SARS), and 3 cases of acquired immune deficiency syndrome (AIDS) in the course of treatment for 1 to 5 days, 500-700 ml per day. RESULTS: For all the 9 SARS cases, their hypoxemia was gradually corrected to normal in 20 minutes' or 4 hours' intravenous drip with oxygen-enriched liquid. Respiratory rate decreased from 29-49 breath/min to 18-22 breath/min, heart rate decreased from 89-145 beats/min to 60-79 beats/min, two faint patients regained consciousness, hypoxemia was redressed, partial pressure of oxygen in artery increased from 56 mm Hg (1 mm Hg=0.133 kPa) to 87 mm Hg, saturation of oxygen increased from 0.89 to 0.96. CONCLUSION: Intravenous drip of the oxygen-enriched liquid effectively helped correct the hypoxemia of SARS and other infectious diseases cases by bypassing the diseased alveoli through which oxygen would not pass into the blood by conventional oxygen inhalation. This therapy of oxygen-enriched liquid infusion could be quite life-saving in the combined treatment for SARS and other infectious diseases.

[Full-length sequence of hepatitis B virus isolated from high incidence hepatocellular carcinoma area-Longan county].

OBJECTIVE: To understand full-length sequence of HBV isolated from high incidence hepatocellular carcinoma area-Longan county, Guangxi. METHODS: The nested polymerase chain reaction (nPCR) was used for amplifying the whole HBV DNA in sera of asymptomatic carriers. The products were sequenced by clone sequencing and homological analysis. RESULTS: This isolate contained 3 215 bases. The genotype was C and the serotype was adw. There were 40 point mutations in polymerase gene which made 11 amino acids change. There were 11,2 and 3 point mutations in PreS1, PreS2 and S gene respectively which made 3,1,1, amino acids change. Six point mutations including the double mutations (nt 1762 A to T, 1764 G to A) were found in X gene leading to 4 amino acids change. There were 13 point mutations in C gene which made 2 amino acids change. No mutation was found in a determinant and Pre C. The isolate was quite close to the isolate from Vietnamese in evolution while far from the genotype C isolates from Shanghai, Beijing and Tibet. CONCLUSION: No special sequence was found in the isolate from high incidence hepatocellular carcinoma area, Longan county, Guangxi.

Pathomorphological study on location and distribution of Kupffer cells in hepatocellular carcinoma.

AIM: To clarify the location and distribution of Kupffer cells in hepatocellular carcinoma (HCC), and to investigate their role in hepatocarcinogenesis. METHODS: Kupffer cells were immunohistochemically stained by streptavadin-peroxidase conjugated method (S-P). The numbers of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues of 48 HCCs were comparatively examined. RESULTS: The mean number of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues was 12.7+/-6.8, 18.1+/-8.2 and 18.9+/-7.9 respectively. The number of Kuppfer cells in cancerous tissues was significantly lower than that in para-cancerous tissues (t=2.423, P<0.05) and adjacent normal liver tissues (t=2.521, P<0.05). As tumor size increased, the number of Kupffer cells in cancerous tissues significantly decreased (F=4.61, P<0.05). Moreover, there was also a significant difference in the number of Kupffer cells among well-differentiated, moderately-differentiated and poorly-differentiated cases(F=4.49, P<0.05). CONCLUSION: This study suggests that decrease of Kupffer cells in HCCs may play an important role in the carcinogenesis of HCC, the number of Kupffer cells in HCC is closely related to the size and differentiation grade of the tumor.

[Early indexes to predict the therapeutic effect of interferon on chronic hepatitis B].

OBJECTIVE: To summarize the clinical changing characters of the clinical markers after interferon treatment in chronic hepatitis B (CHB) and make out practical indexes to predict the effect. METHODS: 150 CHB patients were randomly divided into two groups: therapeutic group (90) and control group (60) in the prospective controlled trial. The levels of endogenous interferon before treatment, interferon antibody at the end of the second month and fourth month after treatment, alanine aminotransferase (ALT) and HBV DNA in the serum were detected. Then the data was analysed to find out indexes for predicting the effect. RESULTS: (1) The clearance rate of HBeAg had no significant difference in age except for 20 - 30 and 30 - 40 (t > 2.331 2, P < 0.01). (2) It was more effective if ALT level was higher than 400 U/L before treatment and it decreased more than 50% two months after treatment. (3) The patients whose HBV DNA was negative (dot hybridization) or less than 10(6) copies/ml before treatment had higher rate of HBeAg clearance. (4) There was no effect on patients whose interferon antibody turned positive at the end of the second month. (5)A predictive method of comprehensive factors was made out, whose sensitivity, specificity, and accuracy were 80%, 100% and 90%, respectively. CONCLUSION: The clinical characters of these Chinese patients are different from those of the westerners and the effects of interferon have close relation to the levels of ALT, HBV DNA and interferon antibody.

Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells.

AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably. METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind III/Not I followed by ligation with pRc/CMV, or BamHI/EcoR I followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSG5UTPL/Flag plasmid vectors with T4 DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were Compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418. RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preS1-preS2S and preS1S encoding genes, determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins. Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen. CONCLUSION: Eight recombinant plasmids expressing S, M, L or preS1S proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells, the vector pRc/CMV is superior to pSG5UTPL/Flag, and pRc/CMV-S and pRc/CMV-MS are the most efficient in the pRc/CMV clones. SP2/0 cells stably expressing HBsAg are established, and may be used as target cells for evaluating the CTL activity of a DNA vaccine in vitro.