Features of human cytomegalovirus UL146 gene in asymptomatic children / 中华微生物学和免疫学杂志

Objective To investigate the polymorphisms of human cytomegalovirus ( HCMV ) UL146 gene in asymptomatic children. Methods Urine samples were collected from 47 asymptomatic chil-dren who were positive for HCMV DNA. PCR was performed to amplify the open reading frame ( ORF) of UL146 gene. Positive bands were sequenced and variations in UL146 gene were analyzed by using bioinfor-matics software. Results Seventeen samples were successfully amplified and sequenced. Variations spread all over the sequence of UL146 gene and the variability in nucleotide and amino acid sequences ranged from 0％ to 42. 5％ and 0％ to 67. 7％ respectively. Compared with the Towne strain, there was diversity in sig-nal sequence and C-terminal region. Phylogenetic analysis indicated that UL146 in the 17 asymptomatic chil-dren belonged to four genotypes, which were G1, G8, G9 and G11. Forms of post-translational modification varied greatly among the four genotypes, while the important functional region of ELRCXC chemokine was highly conservative. Secondary structure prediction showed that random-coli conformation was the predomi-nant structure of active proteins. Isoelectric point ( PI) and molecular weight ( MW) were dissimilar among the four genotypes. Conclusion HCMV UL146 gene in asymptomatic children was hypervariable in both nucleotide sequence and amino acid structure. However, the important functional region was highly con-served. The predominant genotypes of UL146 in these children were G1, G8, G9 and G11, and the geno-type distribution in them showed no significant difference with previous findings in children with symptomatic HCMV infection.

Features of human cytomegalovirus UL146 gene in asymptomatic children / 中华微生物学和免疫学杂志

Objective To investigate the polymorphisms of human cytomegalovirus ( HCMV ) UL146 gene in asymptomatic children. Methods Urine samples were collected from 47 asymptomatic chil-dren who were positive for HCMV DNA. PCR was performed to amplify the open reading frame ( ORF) of UL146 gene. Positive bands were sequenced and variations in UL146 gene were analyzed by using bioinfor-matics software. Results Seventeen samples were successfully amplified and sequenced. Variations spread all over the sequence of UL146 gene and the variability in nucleotide and amino acid sequences ranged from 0％ to 42. 5％ and 0％ to 67. 7％ respectively. Compared with the Towne strain, there was diversity in sig-nal sequence and C-terminal region. Phylogenetic analysis indicated that UL146 in the 17 asymptomatic chil-dren belonged to four genotypes, which were G1, G8, G9 and G11. Forms of post-translational modification varied greatly among the four genotypes, while the important functional region of ELRCXC chemokine was highly conservative. Secondary structure prediction showed that random-coli conformation was the predomi-nant structure of active proteins. Isoelectric point ( PI) and molecular weight ( MW) were dissimilar among the four genotypes. Conclusion HCMV UL146 gene in asymptomatic children was hypervariable in both nucleotide sequence and amino acid structure. However, the important functional region was highly con-served. The predominant genotypes of UL146 in these children were G1, G8, G9 and G11, and the geno-type distribution in them showed no significant difference with previous findings in children with symptomatic HCMV infection.

Detection of the plasma miR-92a expression in systemic lupus erythematosus and its clinic significance / 中华风湿病学杂志

Objective To analyze miR-92a expression and its clinical significance in the plasma of systemic lupus erythematosus (SLE) patients.Methods Plasma samples from 44 SLE patients,16 rheumatoid arthritis (RA) patients and 20 healthy controls were collected.The small RNAs in these plasma samples were isolated and reversely transcribed.Using cel-miR-39 as the external reference,the levels of miR-92a expression were detected by real-time polymerase chain reaction (PCR) method.MiR-92a and cel-miR-39 were analyzed by real-time fluorescence quantitative PCR and agarose gel electrophoresis.The sensitivity and specificity of miR-92a as SLE were analyzed by receiver-operating characteristic (ROC) curve.The correlation between the levels of miR-92a expression and the clinic pathological features of SLE and biological significance of miR-92a expression in SLE were further analyzed by Pearson or Chi-square test.Results Our data indicated that the plasma levels of miR-92a expression was 49.20 (5.33,95.17) in SLE patients,411.30 (320.84,504.69) in healthy controls,and 25.59(11.20,30.54) in RA patients.The difference was significant (x2=40.77,P＜0.01).The area under the ROC curve (AUC) was 0.958 for discriminating between SLE patients and normal subjects and 1.00 for discriminating between RA patients and healthy controls.The levels of miR92a expression cutoff values were set the as 198.59 for healthy control and 85.35 for RA patients,the diagnostic sensitivity and specificity were 93.2％,90％,and 100％,100％,res-pectively.The analysis of the correlation between miR-92a expression and the clinic pathological features of SLE had shown that the levels of plasma miR-92a expressions were much lower in SLE patients with down-regulated complement C3,and up-regulated urea nitrogen,creatinine,LDH,ATH (all P＜0 05).Conclusion Down-regulated miR-92a expression in plasma of SLE may be involved in the SLE disease occurrence or development and could be used as a novel potential diagnostic biomarker for SLE.

Constructing a recombinant adenovirus carrying human cytomegalovirus UL138 gene and evaluating the effects of UL138 gene on the functions of monocytes / 中华微生物学和免疫学杂志

Objective To construct a recombinant adenovirus carrying UL138 gene, which was re-lated to the latent infection of human cytomegalovirus, and to investigate the effects of UL138 gene on the functions of THP-1 mononuclear cells. Methods The recombinant adenovirus expressing the UL138 gene was packaged. The titer of the recombinant adenovirus was determined by calculating 50% tissue culture in-fective dose ( TCID50 ) . THP-1 mononuclear cells were infected with the recombinant adenovirus at different multiplicity of infection (MOI) and the optimal MOI was determined (100 PFU/cell) by observing the ex-pression of green fluorescent protein ( GFP ) . Changes in the expression of proinflammatory cytokines by THP-1 mononuclear cells that was induced by overexpressed UL138 were analyzed by quantitative PCR. The expression of chemokines and their receptors were measured by quantitative PCR array. Results The re-combinant adenovirus carrying the UL138 gene was successfully constructed with a titer of 1×1011 PFU/ml. The rate of THP-1 mononuclear cells that was infected with the recombinant adenovirus was 60% at the MOI of 1 ∶ 100. Results of the RT-PCR analysis and Western blot assay further confirmed that the recombinant adenovirus could infect THP-1 mononuclear cells successfully and the expression of UL138 protein increased gradually over time. The overexpressed UL138 in THP-1 mononuclear cells significantly inhibited the expres-sion of IL-18, IL-1β, IL-6, IL-8 and TNF-α as indicated by the results of quantitative PCR. Results ob-tained from the quantitative PCR array analysis showed that most of the chemokines and their receptors were downregulated in the transfected THP-1 mononuclear cells except for the chemokines of CCL17, CCL21, CCL2 and XCL2 and the receptors of CCR2, CXCR1, CXCR2, CXCR4 and CX3CR1 which were upregulat-ed. Conclusion We successfully constructed the recombinant adenovirus carrying UL138 gene which could be used to infect THP-1 mononuclear cells. Overexpressed UL138 in THP-1 mononuclear cells significantly affected the functions of THP-1 mononuclear cells.

Construction of recombinant yeast expressing PreS2120-146 and hepatitis B surface antigen and evaluation of the immune effects of whole yeast cells / 中华传染病杂志

Objective To construct the recombinant yeast expressing PreS2 120-146-hepatitis B surface antigen (HBsAg),and to evaluate the immune effects of whole yeast cells.Methods PreS2 120-146 and HBsAg gene sequence were optimized according to the yeast cell codon preference,and were recombined and cloned into pPIC3.5K yeast expression vector to construct pPIC3.5K/PreS2 120-146 plasmid.After digested and linearized by Bgk Ⅱ restriction enzyme,pPIC3.5K/PreS2 120-146-HBsAg recombinant plasmid was electrotransformed into GS115 strain to screen PreS2 120-146-HBsAg-recombinant Pichiapastoris .The expression of PreS2 120-146-HBsAg was identified by sodium doclecyl sulfate polyacrylamide gel electrophogesis (SDS-PAGE),Western blot and enzyme linked immunosorbent assay (ELISA)analysis. BALB/c mice were vaccinated by inactivated whole recombinant yeast cells expressing target protein. Specific antibodies to HBsAg were detected by ELISA.Cytotoxic T lymphocyte (CTL)response induced by interferon (IFN)-γ was detected by reverse transcription-polymerase chain reaction (RT-PCR)when immune spleen cells of mice were stimulated by CTL epitope on HBsAg.Independent sample t test was used. Results Data of PCR detection,restriction enzyme digestion and sequencing analysis showed that recombinant pPIC3.5K/PreS2 120-146-HBsAg plasmid was successfully constructed.SDS-PAGE,Western blot and ELISA verified the expression of PreS2 120-146-HBsAg in the lysate of the recombinant Pichiapastoris induced by methanol.Levels of specific anti-HBsAg IgG antibodies produced by inactivated yeast cells vaccinated mice were comparable to purified HBsAg immunization (t =0.946,P =0.381 ). Analysis of HBsAg-specific CTL responses revealed that the level of IFN-γwas significantly higher when the immune spleen cells of mice were stimulated by CTL epitope peptides on HBsAg (t =2.305 ,P =0.044).Conclusions PreS2 120-146-HBsAg target protein is successfully expressed by construction of recombinant Pichiapastoris . The specific humoral and cellular immune responses are induced by recombinant whole yeast cells vaccinated mice.

Human cytomegalovirus infection and its significance in peripheral blood leukocytes of patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To detect the prevalence of human cytomegalovirus (HCMV) in peripheral blood of patients with systemic lupus erthematosus (SLE) and explore its role in the pathogenesis of SLE.Methods HCMV DNA was isolated from the peripheral blood leucocytes (PBLs) of 60 patients with SLE and 111 healthy controls.Nested polymerase chain reaction (nPCR) technology was used to investigate the gene of HCMV glycoprotein gB (UL55) in these specimens.HCMV infections in the PBLs of SLE patients were confirmed by HCMV-UL55 detection.Two-sample t test,nonparametric test,Chi-square test and Fisher probabilities were used to analyze.Results Agarose gel electrophoresis and sequencing analysis showed that established nPCR could specifically detect HCMV-UL55 gene,the HCMV infection rate was significantly higher in patients with SLE than in the healthy controls (P＜0.01).Positive rates of HCMV infection in SLE group and controls were 41.7％ (25/60) and 1.8％ (2/111),respectively.Compared to the SLE patients with HCMV-negative PBLs,the positive rate of Rib-P [26％(9/35) vs 56％(14/25),x2=5.659,P=0.017],the positive rate of direct Coomb's test [37％(13/35) vs 72％(18/25),x2=7.096,P=0.008] and the level of antiβ2GP1 [21.3 (9.9,51.8) U/ml vs 13.6 (5.9,23.1) U/ml,U=2.017,P=0.044] were significantly higher than those in the SLE patients with HCMV-positive PBLs.Compared to the SLE patients with HCMV-negative PBLs,the number of red blood cells [(3.65±0.10)×1012/L vs (3.17±0.17)×1012/L,t=2.574,P=0.013] and lymphocytes [(1.37±0.14)×1012/L vs (0.90±0.13)×1012/L,t=2.456,P=0.017] in peripheral blood and the hemoglobin levels [(110±19) g/L vs (98±5)g/L,t=2.034,P=0.048] of the SLE patients with HCMV-positive decreased significantly.At the same time,the positive rate of hematuria [40％(14/35) vs 72％(18/25),x2=6.000,P=0.014] and 24 h proteinuria [0.80 (0.53,2.37)g vs 0.48 (0.13,1.21)g,U=2.140,P=0.032],which indicated kidney damage were also significantly increased in SLE patients with HCMV-positive PBLs.Conclusion The infection of HCMV in peripheral blood cells may take part in the development of SLE.

Expression and regulatory mechanism of microRNA-155 in the villi of patients with unexplained recurrent spontaneous abortion patients / 中华妇产科杂志

Objective To study the expression and the mechanism of miR-155in the villi of patients with unexplained recurrent spontaneous abortion (URSA).Methods The expression of miR-155 in the villi of 36 cases with URSA (URSA group) and 25 women with normal early pregnancy (control group) were detected by stem-loop real-time reverse transcription (RT) qPCR.Expression of hypoxia inducible factor-1 (HIF-1α),vascular endothelial cell growth factor(VEGF) and micro lymphatic vessel density (MVD) in the villi of were measured by immnohistochemical staining among two groups.Results (1) miR-155 expression:the mean miR-155 expression were 1.456 (0.489,2.459) in URSA group and 2.833 (1.740,3.794) in control group,which reached statistical difference (P ＜0.05).The mean expression of miR-155 of 1.683 (0.902,2.459) in URSA group with abortion times (≤ 3) was significantly higher than 1.229 (0.489,1.719) in URSA group with more than 4 times abortion (P ＜ 0.05).(2) Indexes:the expression of HIF-1α,VEGF and MVD value were 121 ± 12,134 ± 12,36 ± 6 in URSA group and 99 ± 10,109 ± 10,28 ±4 in control group,which reached statistical difference(P ＜ 0.01).The expression of HIF-1α,VEGF and MVD value of 119 ± 12,134 ± 12,35 ± 5 in URSA group with less than 3 times abortion was significantly lower than 128 ± 12,138 ± 12,43 ± 6 in URSA group with more than 4 times abortion (P ＜ 0.01).Conclusions The expression of miR-155 and HIF-1α is topically stimulated by oxygen signal.HIF-1α adjusts the transcription and translation of VEGF,which together involved in placental trophoblast invasion and placental angiogenesis.The low expression of miR-155 could interfere with expression of HIF-1α and VEGF,which might be involved in villous vascular dysplasia in URSA.

differential expression of microRNA-143,145 in endometriosis

Background: Recent studies showed that inappropriate expression of microRNAs [miRNAs] is strongly associated with tumor-related processes in humans [2-9,11-17]

Objective: To understand the changes of miRNAs in endometriosis

Materials and Methods: With real-time RT-PCR, we investigated the miR-143 and miR-145 expression in eutopic [EU, n=2] and ectopic endometrium [EC, n=11] [from women with endometriosis] [as well as EU+EC, n=11], along with the normal endometrium [EN, n=22] [from women without endometriosis, but with leiomyoma]

Results: We did not find that the expression of miR-143 and/or miR-145 in EN or EC changed with menstrual cycle. But our results showed the miR-143 was up-regulated in EC [p=0.000] compared to EN. The miR-143 was also up-regulated in EU, but the difference did not reach statistically significance [p=0.053]. Compared to EU, the expression of miR-143 in EC was up-regulated [p=0.016]. MiR-145 had the similar characteristic to miR-143. The miR-145 was up-regulated in both EU [p=0.004] and EC [p=0.000] in compared to EN group. When compared with EU, the miR-145 in EC was up-regulated [p=0.008]

Conclusion: In conclusion, the miR-143 and miR-145 may play a certain role in the development and progression of endometriosis

Preliminary study of microRNA related to renal interstitial fibrosis in rats / 中华肾脏病杂志

Objective To investigate the expression pattern of microRNA (miRNA) in the kidneys of unilateral ureteral obstruction (UUO) rats and to identify specific miRNA related to renal interstitial fibrosis (RIF).Methods Forty-eight male SD rats were divided into two groups:UUO group and sham-operated (Sham) group.Rats were sacrificed at 3,7 and 14 days after operation.Histologic changes were examined by Masson staining.Forty-eight selected miRNAs were examined by stem-loop real-time qPCR.Results At the 3rd day after operation,obstructed kidneys from operation rats showed mild edema in the interstitium and mononuclear cell infiltration.At the 7th day after operation,focal interstitial fibrosis was observed.At the 14th day after operation,fibrosis became more severe.The Sham kidneys showed no pathological changes.At the 3th day after operation,25 miRNAs were differentially expressed.At the 7th day after operation,24 miRNAs were aberrantly expressed,whereas 21 miRNAs were differentially expressed at the 14th day after operation (P＜0.05).Among these miRNAs,miR-132,miR-192,miR-194,miR-29c and miR-203 were consistently up-regulated or down-regulated in a time-dependent manner after operation.There were significantly correlations between the expression of five miRNAs and severity of tubulointerstitial injury (P＜0.05).Conclusions There are at least 20 miRNAs differentially expressed in the process of RIF induced by UUO.There are significantly correlations between the expression of miR-132,miR-192,miR-194,miR-29c and miR-203 and the severity of tubulointerstitial injury.They may be closely related to RIF.A further study is needed.

Prediction and research on homology of B-cell epitopes of Epstein-Barr virus nuclear antigen-1 / 生物医学工程学杂志

We predict in this paper B-cell epitopes of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and analyze the results matched with the related autoantigens sequence of human. We selected EBV-1 standard strain NA-1 amino acid sequence as the basis. We predicted B-cell dominant epitopes of EBNA-1 with the methods of SOPMA, GOR and HNN, combined with the multi-parameter analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenicity index, polarity and average flexibility. The blastp method was adopted to analyze the matched results between the predicted B-cell epitopes of EBNA-1 and the related autoantigens sequence of human. The results have shown that the possible B-cell dominant epitopes of EBNA-1 were located in the N terminal regions of 16-23, 35-78, 332-337, 340-357, 398-404, 419-432 and 620-637, in which different regions gained higher scores when matched with small nuclear ribonucleoprotein SmB, SmD, ribonucleoprotein SSA, heterogeneous nuclear ribonucleoprotein hnRNP A1, hnRNP G, respectively. It was available to predict B-cell dominant epitopes of EBNA-1 with multiparameter methods and to analyze the same or similar autoantigens sequences of human, which laid a theory foundation for the study of pathogenesis, diagnosis and treatment of autoimmune diseases.

Association of PPARγ2 gene C1431T polymorphism to serum adipokines in women with poly cystic ovary syndrome / 中华内分泌代谢杂志

PPARγ2 gene C1431T polymorphism was assayed by PCR-RFLP in 200 polycystic ovary syndrome( PCOS)patients and 150 normal subjects. Serum adiponectin and leptin levels were determined by ELISA method. Polymorphism of the site might be associated with serum leptin and adiponectin concentrations and thiazolidinedione treatment in women with PCOS ( P<0.05 or P<0.01).

Clinical significance of miR-143 and miR-145 expression in gastric gastrointestinal stromal tumor / 中华普通外科杂志

Objective To evaluate the role of miR-143, miR-145 in the development of gastric gastrointestinal stromal tumor. Methods The expression levels of miR-143 and miR-145 in 21 cases of gastric gastrointestinal stromal tumor and the matched non-tumor adjacent tissue specimens were examined by stem-loop real-time RT-PCR, and its correlation with clinicopathologic features of gastric gastrointestinal stromal tumor were analyzed. Results Expression level of miR-145 were significantly higher in tumor than adjacent normal tissues (P＜0.01 ) and that with mitotic count ≥ 5/50HPF cases was significantly lower than that with mitotic count ＜5/50HPF cases (P=0.02). miR-145 expression in huge tumor (＞10 cm)was significantly lower than that in the large tumor (5～10 cm) and small tumor (2～5 cm) (P=0.048).By Fletcher risk stratification system, miR-145 expression in high-risk cases was significantly lower than that in the intermediate-risk and low-risk cases (P=0.048). While the expression of miR-145 in low-risk group was significantly different compared to that in intermediate-risk group and high-risk group (P=0.01).There was no difference between the expressions of miR-143 in tumor and that in normal tissue(P=0.06).Conclusion In gastric gastrointestinal stromal tumor, MiR-145 expression is significantly higher in tumor than adjacent normal tissues. miR-145 is closely associated with tumor size. mitotic counts and Fletcher risk stratification system.

Research on homology and cross reaction characteristics of human papillomavirus L2 N-terminal protein / 中华微生物学和免疫学杂志

Objective To research the homology and cross reaction characteristics of human papillomavirus(HPV)16 type L2 N-terminal(1-200)protein in clinical common HPV infection types.Methods The amino acid sequences of the common HPV infection types(6,11,16,18 ,etc.)were blasted and it was found that 1-200 N-terminal sequence of L2 protein was highly homologous.The gene of HPV16 L2(1-200)was amplificated from tissue sample of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2(1-200).After sequencing identification,the recombinant plasmid was tranformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein containing HPV16 L2(1-200)was expressed and analyzed by both SDS-PAGE and Western blot.Furthermore,the specific binding capacity of the fusion protein to the HPV 6,11,16 and 18 DNA positive patient serums were analyzed by Western blot.The fusion protein was purified with Ni-NTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG of 98 condyloma acuminatum patients,135 cervix cancer patients and 96 healthy control subjects were detected respectively by indirect ELISA.Results After comparing the amino acid sequences of the common HPV infection types(HPV6,11,16,18,etc.),We found that the homology of HPV L2(1-200)reached 52.7%-74.3%.The recombinant plasmid PGEX-4T-1-HPV 16 L2(1-200)was constructed successfully.Highly expressed HPV16 L2(1-200)fusion protein was obtained and the expression level was account for up to about 22.6% of total bacterial protein.The relative molecular mass(Mr)of the fusion protein is about 49×103,which matches up to the expected Mr Meanwhile,the serums of HPV 6,11,16,18 DNA positive patients were used as the first antibody and the specific band was detected respectively at about 49 × 103 by Western blot.Indirect ELISA showed that the A490 values of the specific IgG of condyloma acuminatum group,cervical cancer group and healthy control subjects were 0.753 ± 0.262,0.756 ± 0.274 and 0.178 ± 0.157 with the positive rate were 89.8%,88.9% and 9.4% respectively.There was no significance of the specific IgG between condyloma acuminatum group and cervical cancer group(P＞0.05),but it was significant among the three groups(P＜0.001).Conclusion The N-terminal 1-200 amino acids of HPV L2 has high homology and there exits cross reaction among the most common HPV infection types.