Development and application of a safe SARS-CoV neutralization assay based on lentiviral vectors pseudotyped with SARS-CoV spike protein / 病毒学报

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike protein (S) is a major target for neutralizing antibody. To develop and apply a safe neutralization assay for SARS-CoV, lentiviral SARS-CoV S pseudotypes had been constructed based on a three plasmid system, which contained pVRC8304 (harboring codon optimized full-length SARS-CoV S protein), pCMV delta 8. 2 (HIV-1 gag/pol construct) and pHR'CMV EGFP (the green fluorescent protein reporter construct). The pseudo-typed lentiviral particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for SARS-CoV neutralizing antibody. We used this assay to determine the titers of the neutralizing antibodies (Nabs) in serum samples from mice immunized with various rVVs expressing different S fragments of SARS-CoV. The serum antibodies derived from S and various segments of S1 region neutralized SARS-CoV in vitro. No cross-neutralization occurred with the goat antiserum prepared with inactivated HCoV-OC43 or HCoV-229E. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live SARS-CoV. Because the pseudotype assay does not require handling live SARS virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.

Acute toxicity and immunoprotection of recombinant apxI toxin of Actinobacillus pleuropneumoniae in mice / 生物工程学报

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.

Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line (2BS) induced by silica.</p><p><b>METHODS</b>Recombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control.</p><p><b>RESULTS</b>During the progress of the malignant transformation of 2BS cells induced by silica, cyclinD1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively), and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% (the growth rate of malignant transformed cells induced by silica was 100%), doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously.</p><p><b>CONCLUSION</b>CyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.</p>

Role of cyclin D1 in carcinogenesis of human cells induced by quartz / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the role of cyclin D1 in malignant transformation of human embryonic lung diploid fibroblasts (HELF) induced by quartz.</p><p><b>METHODS</b>pXJ41-cyclin D1 expressing sense and antisense cyclin D1 RNA were transinfected into malignant transformed HELF induced by quartz with DNA recombination and gene transduction. The expression of cyclin D1 was detected with hybridization in situ and immunohistochemistry methods to analyze changes in cell growth, double multiplication time, distribution of cell cycles, colony forming ability on soft agar, etc., before and after cyclin D1 transduction.</p><p><b>RESULTS</b>During the process of malignant transformation of HELF induced by quartz, cyclin D1 gene was overexpressed. Antisense pXJ41-cyclin D1 RNA could suppress the growth and proliferation of malignant transformed cells induced by quartz. Growth speed of antisense pXJ41-cyclin D1 transinfected cells decreased by 58.69% on the 8th day in culture, as compared to malignant transformed cells induced by quartz, and its double multiplication time prolonged from 21.0 h to 31.4 h. Antisense cyclin D1 RNA led to cell cycle arrest, resulting in lengthened G1 phase (proportion of cells in phase G1 increased to 52.7% from 45.1% and that of cells in phase S decreased to 33.1% from 40.3%). Colony forming rate reduced significantly and size of colony became smaller.</p><p><b>CONCLUSIONS</b>Abnormal expression of cyclin D1 in cells related to their malignant transformation induced by quartz. Highly expressed cyclin D1 could play an important role in maintaining the transformed phenotype of malignant cells.</p>

The role of cycline dependent kinase 4 in the malignant transformation induced by silica / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of cycline dependent kinase 4 (CDK4) in the malignant transformation of human fetal lung diploid fibroblast cell (2BS) induced by silica.</p><p><b>METHODS</b>Recombination vectors with antisense pXJ41-CDK4 were constructed, and then were transfected into the malignant transformed cells induced by silica. In situ hybridization and immunohistochemistry were used to analyze the expression of CDK4. Cell growth curve, doubling time, cell cycle distribution and the growth capacities on soft agar were analyzed before and after antisense CDK4 RNA was transferred into malignant transformed cells induced by silica.</p><p><b>RESULTS</b>During the malignant transformation of 2BS cells induced by silica, CDK4 gene was overexpressed. Antisense pXJ41-CDK4 transduction suppressed CDK4 gene expression in the antisense pXJ41-CDK4 transfected cells. Antisense CDK4 RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase increased from 45.1% to 58.0%), and eventually attenuated the proliferation of malignant transformed cells induced by silica. At the 8th day, the suppression rates decreased by 77.43%. The doubling time prolonged from 21.0 h to 42.7 h. The growth capacities on soft agar of cells transfected by antisense pXJ41-CDK4 were decreased.</p><p><b>CONCLUSION</b>CDK4 might play an important role in maintaining the transformed phenotype of the cancer cells.</p>