Community Impact Assessment(EIA) provides an objective basis for judging the correct application of the non-custodial sentence,which reduces the risk of application of the Community Correction,and are conducive to the early involvement of the community correction agencies.In practice,legislative lag,lack of procedural safeguards and other factors affect the judicial and administrative authorities in carrying out the work,and the community impact assessment is difficult to play its due role.There is an urgent need for an in-depth analysis of and theoretical and practical resolution to this problem.

Objective To express the excellular domain of glycoprotein D(gD) of herpes simplex virus type 2(HSV-2) in mammalian cells and analyze the immunologic activity of expressed product.Methods The gDt gene sequence encoding excellular domain of gD of HSV-2 G strain was chemically synthesized and inserted into vector pCEP4.The constructed recombinant plasmid pCEP4-gDt,with a His label at N-terminus,was transfected to HEK293 cells for expression.The expressed protein was purified by nickel ion column affinity chromatography and tested for antigenicity by ELISA.Polyclonal antisera were prepared by immunizing mice with the purified recombinant protein and determined for titer by ELISA.Results PCR,restriction analysis and DNA sequencing proved that recombinant plasmid pCEP4-gDt was constructed correctly.Western blot showed a target protein band with relative molecular mass of about 46 000.The purified recombinant protein,at a concentration of about 45 μg/ml,showed good antigenicity as proved by ELISA,which induced specific antibody titer of 5 × 103 in the sera of mice 5 weeks after immunization.Conclusion The excellular domain of gD of HSV-2 was expressed in mammalian cells,and showed good antigenicity and immunogenicity,which laid a foundation of preparation of recombinant subunit vaccine against HSV.

To express and purify herpes simplex virus type I(HSV I)glycoprotein B(gB)in eukaryote cells with the purpose of analyzing the antigenicity and immunogenicity.At the beginning,the extracellular domain fragment gene of gD1 was synthesized by chemical method and cloned into eukaryotic expression vector pCEP4 to construct the recombinant plasmid pCEP4-gD1.After transfection of HEK293 cells with the recombinant plasmid,the expressed protein was characterized by Western blot and purified through Ni affinity chromatography.Then antigenicity of the protein was detected by ELISA.Finally,the purified protein was used to immunize Kunming mice in 1,3,5 weeks respectively,and antiserum were collected in 3,5 and 7 weeks.The antibody titer was detected by indirect ELISA for immunogenicity analysis.Gene sequencing analysis demonstrates that the recombinant plasmid pCEP4-gB1 was constructed successfully.Western blot analysis indicated one major protein band,which molecular weight is approximate 85 kDa corresponding to the truncated forms of gB1 protein,was observed.In addition,ELISA detection showed that expressed gB1 has good antigenicity.After the third immunization,antibody titer of the mouse anti-gB1 was 5×103.The successful expression of the recombinant protein gD1,which can induce humoral immune response,lays a foundation for serological diagnosis and vaccine study of HSV.

Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.

In order to improve the soluble expression of recombinant Hepatitis C virus RNA-dependent RNA polymerase(RdRp) in E.coli,several factors influencing RdRp expression level and solubility were optimized by using single factor tests.The expressed RdRp was detected by SDS-PAGE.The results showed that amplification cultural time,IPTG concentration,induction temperature and induction time have significant effects on solubility of the expressed RdRp.The best condition for expressing soluble RdRp was obtained,when OD600 of the culture medium reached about 1.6,and the engineered E.coli was induced with 0.20mmol/L IPTG at 25℃ for 16h.This study has established the foundation for assaying RdRp enzymatic activity in vitro and screening the anti-HCV drugs targeting RdRp.