Detection of human papilloma virus 16/18 by the quantitative fluorescent PCR / 中华微生物学和免疫学杂志

Objective To study the relation between the HPV6/18 virus infection and the development of pathological changes of cervix. Methods The number of HPV16/18 DNA copies and the expression rate of HPV16/18 E7 mRNA in the pathological cervix were examined by the quantitative fluorescent PCR combined with pathological diagnosis and immunohistochemistry staining. Results The HPV16 infection rates in chronic cervicitis group were much lower (7.4%) than that in the cervical intraepithelial neoplasia (CIN) groups and the cervical cancer group (69.6% and 72.7%), respectively. Statistical analysis showed that the difference of HPV16 DNA copies was not significant between the chronic cervicitis group and CIN groups. In contrast to the above mentioned result, the number of HPV DNA copies between the CIN groups and the cervical cancer group was significantly different. The HPV16 E7 gene expression rates in CIN Ⅰ, Ⅱ, Ⅲ and cervical cancer groups were 0,37.5%,42.9%,63.6%, respectively. Conclusion Ins more common than that with HPV18. The number of HPV16 DNA copies in cervical cancer tissues is markedly higher than that in CIN Ⅱ, Ⅲ groups. The HPV16 E7 mRNA expression is significantly increased in the cervical cancer, and it is more closely correlated to this pathological changes. The quantitative fluorescent PCR can be used to reflect the activity of HPV, and it is a useful method for the screening examination of HPV and for the early diagnosis and treatment of cervical caner.

Temporal expression of HCMV IE1 and pp65 in human glioma U(251) cells / 中南大学学报(医学版)

OBJECTIVE@#To determine whether U(251) cells are permissive for human cytomegalovirus (HCMV), and to investigate the characteristics of temporal expression of proteins IE1 and pp65.@*METHODS@#U(251) cells were infected with HCMV, and then the cells were observed under the transmission electronic microscope, and the viral nucleic acid was detected by PCR, and the expression levels of IE1 and pp65 were analyzed by immunohistochemical assay with anti-IE1 monoclonal antibody and anti-pp65 monoclonal antibody at various time spost infection.@*RESULTS@#Morphological changes of the infected cells appeared under the transmission electron microscope. The viral nucleic acid was detected successfully by PCR. The expression of IE1 was detected firstly at 4h post infection, and reached a peak within 14h, and then decreased. The incoming pp65 was detected at 1h, the low expression levels of pp65 were detected firstly at 4h, and they could remain relatively constant through 96 h, but the maximum expression occurred at 120 h.@*CONCLUSION@#Human glioma U(251) cells are permissive for HCMV, the temporal cascade of HCMV gene expression can be observed in the infected U(251) cells, but it is delayed obviously in the human fibroblast.

Cloning and expression of the succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum Chinese strain in E. coli / 中南大学学报(医学版)

OBJECTIVE@#To clone the full-length gene encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) Chinese strain and express it in Escherichia coli.@*METHODS@#According to the published incomplete EST (BU804141) of SjSDISP and the sequence of multiclone sites of lambda gt11 vector, 2 pairs of primers were designed and synthesized. Then the 3' and 5'ends of the EST of the SjSDISP from adult Schistosoma japonicum cDNA library were amplified by anchored PCR. After sequencing, a full-length cDNA sequence of the SjSDISP was obtained, and then it was cloned into prokaryotic expression vector pGEX-4T-1. Identified by agarosed gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for the expression under the temperature-dependent condition and Western blot analysis.@*RESULTS@#A 1,071 bp sequence was obtained. Sequence analysis showed that the fragment contained a complete open reading frame (ORF), encoding 278 amino acid residues. This target fragment was cloned into the prokaryotic expression vector pGEX-4T-1, and expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of the expressed fusion recombinant product was 56 kD. Western blot showed that the recombinant protein was recognized by polyclonal rabbit antiserum immunized with Schistosoma japonicum adult worm antigen.@*CONCLUSION@#Cloning of the full-length gene encoding SjSDISP and its bacterial expression were successfully done.

Cloning,Expression and Immunization of The Hypoxanthine-guanine Phosphoribosyltransferase for Schistosoma japonicum Chinese Strain / 生物化学与生物物理进展

A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library ?gt11 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E.coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partial protection vaccine candidate.

Effect of HCMV on p38MAPK, apoptosis and cell cycle of human glioma U251 cells / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the changes of p38MAPK expressions, the frequency of apoptosis and the distribution of cell cycle of hunan Glioma U251 cells after HCMV infection.</p><p><b>METHODS</b>The expression of total p38 (both phosphorylated and nonphosphorylated p38) and phosphorylated p38 in U251 cells were detected by Western blotting at 15 min, 30 min, 1 h, 6 h, 10 h, 16 h, 24 h, 36 h and 48 h after HCMV infection. The apoptosis percentage and the cell cycle distribution of U251 cells at 2 d, 5 d and 7 d after HCMV infection were detected by flow cytometry (FCM).</p><p><b>RESULTS</b>The results of Western blotting demonstrated that a strong increase in phosphorylated p38 was detected from 6 h to 10 h after HCMV infection, with mean gray scales 186.33 +/- 7.51 (t = 5.37, P < 0.01) and 188.00 +/- 7.02 (t = 5.26, P < 0.01 for all) at 6 h and 10 h, respectively, and p38 phosphorylation decreased to the basic level at 16 h after HCMV infection. But the overall levels of p38 protein were not significantly altered during the course of infection. FCM analysis showed that HCMV could significantly increase the apoptotic rates of U251 cells compared with controls (t = 10.84, P < 0.01), and the apoptotic percentages of the cells reached to peak [(10.18 +/- 1.24)%] at 5 d after HCMV infection. The data of FCM showed that HCMV could decrease the number of U251 cells in G1 phase and arrest the cells in S and G2 phase. The numbers of G1 phase U251 cells were significantly lowered to (56.50 +/- 2.57)% (t = 26.45, P < 0.01), (62.33 +/- 2.64)% (t = 21.20, P < 0.01) and (67.45 +/- 4.44)% (t = 10.61, P < 0.01), respectively at 2 d, 5 d and 7 d after infection.</p><p><b>CONCLUSION</b>HCMV could activate p38MAPK pathway and trigger apoptosis and interfere cell cycle in U251 cells.</p>

Cloning, Expression and Immunization of The Hypoxanthine-guanine Phosphoribosyltransferase for Schistosoma japonicum Chinese Strain / 生物化学与生物物理进展

A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the '3' and 5' ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library λgt1 1 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase ofSchistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E. coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partia1 protection vaccine candidate.

Expression of HOXB9 Gene in Hela Cells,Mocoy Cells, SP2/0 Cells and U251 Cells / 中国医师杂志

Objective To study the expression of HOXB9 gene in Hela cells,Mocoy cells, SP2/0 cells and U251 cells. Methods The expression of HOXB9 gene was detected by semi-quantitative RT-PCR method. Results Hela cell and U251 cell expressed HOXB9 gene, which SP2/0 cell and Mocoy cell didn't express it. Conclusion The expression of HOXB9 gene was different in different cells.

Establishment of Mathematic Model of the Correlativity of HCMV Infection Between Mothers and Their Newborns / 中国医师杂志

Objective To establish a mathematic model of correlativity of human cytomegalovirus(HCMV) infection between mothers and their newborns.Method The plasma HCMV-IgM content,HCMV-DNA levels in plasm and peripheral blood mononuclear cells (PBMCs) were assayed with ELISA and PCR technique.Results There was a evident correlation between the two generation,and there was largest correlativity between HCMV-DNA level of mother's plasma and HCMV congenital infection rate,its spearman rank correlative coefficient was 0 7018(P