Cloning and efficient prokaryotic expression of soluble stage-specific antigen cC1 from Cysticercus cellulosae / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.</p><p><b>METHODS</b>The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>The fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography.</p><p><b>CONCLUSION</b>The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.</p>

Study on the probability of hepatitis B virus infection at public service places / 中华流行病学杂志

Objective To provide data for the control and prevention of hepatitis B and HBV surface antigen(HBsAg)status among the appliances and practitioners working in the public service places.Methods 63 beauty parlors,barber shops and bathing centers selected under stratified randomization sampling method and 682 workers were investigated through questionnaire.HBsAg from the appliances of the public service places and employee was detected by RIA.Results Two main sanitizing modes that including alcohol cleaning(34.60%)and ultraviolet light disinfection(30.79%)were used.The rates of testing on HBsAg among the appliances were 2.13% at the public service places,and were 0.63%,2.67% and 3.70% in large-.medium-and small-sized appliances respectively.The rate of testing on HBsAg on large-,medium-and small-sized appliances were statistically different(χ2=6.68,P＜0.05).The positive rates of HBsAg on the appliances of beauty parlors,barbering shops and footbath inns were 2.97%,0.61% and 3.42% respectively.People working in different service sites had different rates of HBsAg:those who worked at the‘acne needle'and the forceps were 5.13% and 4.17%.The positive rate of HBsAg among the workers in the public service places was 7.13%.The rates of HBsAg among the workers in large-,medium-and small-sized public service places were 7.34%,8.33% and 2.94% respectively.The rates of HBsAg among the workers in beauty parlors,barbering shops,footbath inns and bathing centers were 9.01%,6.37%,4.35% and 7.29% respectively.HBsAg positive rates were different among the workers working at different service sites:13.33% at tattoo business.12.68% in pedicures workers and 8.03% in massagists.Conclusion It is important to improve the sanitizing management of the appliances used in the public service places and to improve the knowledge,attitude,as well as practice of vaccination on hepatitis B among those populations.

Influence of immunization dose schemes on immunoprotective response to recombinant signaling protein 14-3-3 of Schistosoma japonicum / 中华预防医学杂志

<p><b>OBJECTIVE</b>To discuss the optimal immunization dose by observing the immunoprotective effects of different doses of recombinant Schistosoma japonicum (Chinese strain) signaling protein 14-3-3 (rSj14-3-3).</p><p><b>METHODS</b>Sj14-3-3 gene was amplified by reverse transcriptase PCR (RT-PCR), subcloned into prokaryotic expression vector pET28a, then transformed into E.coli to express by inducing. Purified rSj14-3-3 was prepared through SDS polyacrylamide gel electrophoresis (SDS-PAGE), electroelution, dialysis, then BALB/c mice were divided into 5 groups and immunized in rSj14-3-3 protein followed by challenging infection (the 1st, 2nd, and 3rd groups were immunized in 50 microg, 100 microg and 300 microg antigen, respectively. The 4th, 5th groups were immunized in Freund's adjuvant and normal saline controls). After 6 weeks of challenging infection, the mice were killed and the worm and egg reduction rates were calculated. And the mice sera in different time were taken to examine the specific anti-Sj14-3-3 IgG.</p><p><b>RESULTS</b>rSj14-3-3 protein was expressed successfully. After immunizing and challenging, worm reduction was found to be 28.20% in the 1st group, 43.10% in the 2nd group, 40.00% in the 3rd group, respectively. Number of eggs in liver tissue was reduced by 41.80%, 57.50%, 55.70%, respectively. Compared the results of the tested groups to the controls, the differences were of significance by t-test (worm reduction rate: t = 6.8 in the 1st group, t = 8.7 in the 2nd group, t = 7.3 in the 3rd group, P < 0.01 in all tested groups. Egg reduction rate at the group's number above: t = 11.23, t = 11.54, t = 7.99, P < 0.01 in all tested groups). As compared the results between the tested groups by chi(2), the differences were of significance between the 1st and the 2nd groups (worm reduction rate: chi(2) = 8.96, P < 0.05; egg reduction rate: chi(2) = 15.69, P < 0.05), between the 1st and the 3rd groups, the differences were also of significance (worm reduction rate: chi(2) = 6.52, P < 0.05; egg reduction rate: chi(2) = 12.52, P < 0.05). The difference was not of significance between the 2nd and the 3rd groups (worm reduction rate: chi(2) = 1.20, P > 0.05; egg reduction rate: chi(2) = 0.93, P > 0.05). In all tested groups, total anti-Sj14-3-3 specific IgG rose markedly. IgG(1) and IgG(2a) subtypes were high, but IgG(2b) and IgG(3) were near the background in four subtypes tested.</p><p><b>CONCLUSION</b>Immunoprotection of rSj14-3-3 should have some relations with immunization dose, and the protection obtained from immunizing mice by using 100 microg antigen was the best.</p>

Studies on the aberrant methylation of 14-3-3 sigma gene in human breast cancer / 中华检验医学杂志

Objective To observe the methylation and expression of 14-3-3 sigma gene in human breast cancer.Methods 40 breast cancer tissues and 18 mammary gland tissues with benign lesions were analyzed by methylation specific PCR(MSP),RT-PCR,and Western-blot(WB)so as to detect the methylation status and expression of 14-3-3 sigma mRNA or protein.Results Methylation of 14-3-3 sigma gene was detectable in 85%(34/40)of patients with breast cancers.RT-PCR showed negative in 12.5% of breast cancers(5/40),WB also indicated that 14-3-3 sigma was not detected in 32 of 40 breast carcinomas (80%).Furthermore,both RT-PCR and WB were negative in 30 of 34 positive cases by MSP.While methylation of 14-3-3 sigma was not detectable and its expression was demonstrated by RT-PCR and WB among 18 cases of benign breast diseases.These evidences proposed that methylation of 14-3-3 sigma gene had great relevance with its silence.Conclusion Methylation and loss of expression in 14-3-3 sigma gene were high frequent events in breast cancers.And methylation of 14-3-3 sigma gene might be related to its loss.