ABSTRACT
Objective:To express the glycoprotein D of herpes simplex virus type 2 (gD2) in the insect cells,and to determine its immunogenicity.Methods:HSV-2 genome was used as the template for amplification of gD2 extracellular domain fragment gene by PCR.The PCR product was inserted into the vector Bacmind,and the constructed recombinant plasmid gD2-Bacmind was transfected into the sf9 cells to package the recombinant baculovirus.The Sf9 cells were infected by recombinant baculovirus seed derived from the forth passage(P4),the titer of P4 recombinant baculovirus was detected by a plaque assay and the expression of recombinant protein gD2 was determined by Western blotting method.The supernatant of infected cells was collected and purified by Ni-NTA affinity chromatography to obtain the target protein gD2,the purified gD2 protein was used to immunize the BALB/c mice in 0, 2, 4 weeks (gD2 group),and PBS was used as negative control(PBS group);the titers of gD2 specific IgG in serum were detected by ELISA assay.Results: The PCR analysis and sequencing results proved that gD2-Bacmind was constructed correctly.The titer of recombinant baculovirus was 2.0×109 pfu·mL-1,the purified gD2 was about 37 000 with expectation,the percentage of gD2 in total protein was 90%.The average value of Log10 of titer of gD2 specific IgG in serum detected by ELISA assay in gD2 group at the sixth week was 4.34,and there was significant difference compared with PBS group(P<0.01).Conclusion: The gD2 expressed by insect-baculovirus expression vector system has the immunogenicity and can be selected as candidate protein for HSV-2 vaccine.
ABSTRACT
Objective To investigate infection rate of human papillomavirus as well as the correlation between cervical precancerous lesions and co-infection of human HPV,herpes simple virus-2 (HSV-2) and cytomegalovirus (CMV) in Chinese women of childbearing age in Kunming,Yunnan province.Methods A total of 2128 women (18-24,25-34,35-49 years of age),who had healthy care examination in our institute from January 2010 to March 2011,were selected prospectively in this study.The infection of HPV,HSV-2 and CMV were detected by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) and cervical precancerous lesions were determined by the ThinPrep liquid-based cytology test (TCT).Results The overall infection rates of high risk HPV (HR-HPV),HSV-2,CMV were 11.04％(235/2128),3.52％ (75/2128) and 5.26％ (112/2128),respectively.The HR-HPV infection rates in groups of Negative for Intraepithelial Lesion or Malignancy (NILM),Atypical Squamous Cells of Undetermined Significance (ASCUS),Low grade Squamous Intraepithelial Lesion (LSIL),High grade Squamous Intraepithelial Lesion(HSIL),and Squamous Cell Carcinoma (SCC) were 4.29％ (82/1912),55.93％ (66/118),84.62％ (44/52),93.19％ (41/44) and 2/2,respectively.HR-HPV infection rates was increased with the development of cervical lesion (r =0.644,P =0.000).No significant difference on the infection rates of HR-HPV and HSV-2 was identified between different age groups (x2 =2.979,P =0.226; x2 =0.798,P =0.671).The peak age groups for CMV infection (7.62％) were 18 to 24 years old and the infection rates of CMV decrease with age.No significant difference of HSV-2 and HR-HPV coinfection was found between the TCT-abnormal (3.24％,7/216) and control groups (2.41％,46/1912,x2 =0.557,P=0.455),and no relationship was found between HSV-2 and HR-HPV infection groups (OR =0.56,95％ CI:0.17-1.82).The infection of HR-HPV were related significantly with CMV infection (OR =3.14,95％ CI:1.25-7.86).Conclusion HR-HPV infection appears to be the key risk factor for cervical cancer and synergistic interaction may occur between CMV and HPV infections in the development of cervical lesion.
ABSTRACT
Objective To compare the validity of four commercially available ELISA kits for detecting HSV-2 type-specific IgG antibodies. Methods A total of 125 serum specimens were collected from 105 patients with genital ulcers and 20 normal individuals without history of STDs. Four ELISA kits which are commercially available in China for the detection of HSV-2 type-specific IgG antibodies were selected for the evaluation. Western blot assay was used as the gold standard. Results Based on the results of detection by Western blot assay, the sensitivity and specificity of these ELISA kits including home-made 1, home-made 2, improted 1 and improted 2 were 13.1% and 98.4%, 7.5% and 100%, 100% and 11.1%, 87.7% and 96.7%, respectively. The areas under the ROC curve of three kits including home-made 1, imported 1 and imported 2 were 0.885 (0.822 - 0.948), 0.852 (0.747 - 0.902), 0.947 (0.950 - 0.998), respectively. Conclusions The results of imported 2 are well consistent with those of Western blot, while the results of other 3 kits are poorly consistent with those of Western blot. It is also indicated that the commercially available ELISA kits for detecting HSV-2 type-specific antibodies should be re-evaluated in terms of their validity befor being applied for the clinical diagnosis as well as laboratory research.