OSI-027 inhibits the tumorigenesis of colon cancer through mediation of c-Myc/FOXO3a/PUMA axis.

Colon cancer is a gastrointestinal malignancy that is one of the leading causes of tumor-associated deaths. It has been reported that the mammalian target of rapamycin (mTOR) can lead to the progression of colon cancer. However, the mechanism by which mTOR inhibitor (OSI-027) mediates the tumorigenesis of colon cancer remains largely unknown. Cell function of colon cancer was investigated by cell counting kit-8 flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In addition, quantitative real-time polymerase chain reaction and Western blot were used to investigate the mechanism underlying the function of OSI-027 in colon cancer. OSI-027 dose-dependently reduced colon cancer cell viability by inducing cell apoptosis. In addition, OSI-027 induced the apoptosis of colon cancer cells via upregulation of PUMA. OSI-027 promoted the expression of PUMA by activation of forkhead box protein O3a (FOXO3a), and c-Myc knockdown partially increased FOXO3a and PUMA levels. Moreover, OSI-027 attenuated the tumor growth of colon cancer through the mediation of the mTOR/c-Myc/FOXO3a axis. OSI-027 attenuates colon cancer progression through the mediation of the c-Myc/FOXO3a/PUMA axis. Thereby, this study might shed new insights on exploring the strategies against colon cancer.

Highly sensitive detection of cancer-related genes based on complete fluorescence restoration of a molecular beacon with a functional overhang.

The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

A dual molecular beacon approach for fast detection of Mycobacterium tuberculosis.

The main objectives of this study were to assess a dual molecular beacon approach for fast detection of Mycobacterium tuberculosis (MT). MT beacon (Tb-B) was designed to target the unique IS6110 (114 bp) and rpoB (215 bp) fragment of the MT (H37Ra) genome, and the two fragments were inserted into the PMD-19T vector after purification, by PCR and sequencing, to construct plasmids. Different dilutions of positive plasmid standards were used for dual molecular beacon RT-PCR of rpoB and IS6110, and standard curves were established.The results show that the dual molecular beacon of rpoB and IS6110 detecting MT was stable (CV is 1.91-2.68 %) with a high amplification efficiency (95.6 %). In addition, the strains of non MT did not generate fluorescence signals, while strains of MT did, indicating that the primers and molecular beacons were specific, and only MT complex was amplified. The linear range was wide (10(3)-10(11) copies/mL), and clinical specimens presenting different bacterial counts can be detected.

[SNP-chip technology for identification of origins for prenatally detected marker chromosomes].

OBJECTIVE: To determine the origin of 1 prenatally detected small supernumerary marker chromosome (sSMC) using SNP-chip technology, and to deduce the underlying mechanism. METHODS: The fetal sample was subjected to karyotype analysis. The identified sSMC was subjected to genom wide scan using a SNP microarray chip. The results were validated with fluorescence in situ hybridization (FISH). RESULTS: The karyotype of the fetus was determined as 46, X, +mar, which was verified by SNP microarray chip analysis as Yp11.2-11.3 duplication, along with loss of Yq11.2 region, FISH analysis has confirmed that the sSMC has derived from the Y chromosome. CONCLUSION: The karyotype of the fetus was determined as 46, X, idic(Y) (pter→ p11.2::11.2→ pter). Regional deletion of Yq11.2 has been associated with male azoospermia. SNP chip analysis can exclude minor deletions and duplications with a size of more than 1 Mb, which may be applied for verifying difficult cases as well as microdeletion and duplication syndromes upon prenatal diagnosis.

[Characterization of two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss both carrying a mitochondrial 12S rRNA 1494C>T mutation].

OBJECTIVE: To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations, haplotypes, GJB2 gene mutations on phenotype of 1494C>T mutation, and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss. METHODS: Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected. The two probands and their family members underwent clinical, genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene. RESULTS: Clinical evaluation revealed wide range of severity, age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families, for which the penetrance of hearing loss was respectively 42.9% and 28.6% when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss were 14.3% and 14.3%. Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation, in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c, respectively. CONCLUSION: Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families. Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression. However, aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families. The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation. The 1494C>T mutation seems to have occurred sporadically through evolution.

[Modifier factors influencing the phenotypic manifestation of the deafness associated mitochondrial DNA mutations].

Mutations in the mitochondrial DNA have been found to be one of the most important causes of sensorineural hearing loss. In particular, these mutations often occur in the mitochondrial 12S rRNA and tRNA genes. Of these, the homoplasmic A1555G and C1494T mutations in the 12S rRNA have been associated with both aminoglycoside induced and nonsyndromic hearing impairment in many families worldwide. Children carrying the A1555G or C1494T mutation are susceptible to the exposure of ototoxic drugs, thereby inducing or worsening hearing loss. Individuals harboring A1555G or C1494T mutation can also develop hearing loss even in the absence of aminoglycoside exposure. However, matrilineal relatives of intra-families or inter-families carrying the A1555G or C1494T mutation exhibit a wide range of severity, age-at-onset, and audiometric configuration of hearing impairment. These indicate that the A1555G or C1494T mutation is a primary factor underlying the development of deafness but insufficient to produce the clinical phenotype.Thus, other modifier factors, such as aminoglycoside(s), mitochondria l DNA haplotype(s) or nuclear modifier gene(s), play a role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA A1555G or C1494T mutation. In this review, we summarize the modifier factors for the phenotypic expression of deafness-associated 12S rRNA A1555G and C1494T mutations and propose the molecular pathogenetic mechanism of maternally inherited deafness.

[No association of K469E polymorphism of intercellular adhesion molecule-1 with rheumatoid arthritis].

OBJECTIVE: To investigate the association of the intercellular adhesion molecule-1 gene (ICAM-1) K469E polymorphism and rheumatoid arthritis (RA). METHODS: Two hundred and seventy-five patients with RA and 254 healthy individuals were collected and enrolled in the study. The K469E polymorphism of ICAM-1 gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The genotype frequencies of KK, KE and EE of K469E polymorphism were 0.535,0.411 and 0.054 respectively in the RA patients, and 0.512,0.437 and 0.051 respectively in the healthy individuals, and there was no significant difference between the two groups (chi² = 0.371, P = 0.831). The frequencies of the K469 allele were 0.74 and 0.73 in the RA patients and the controls respectively (chi² = 0.127, P = 0.721, OR = 1.051, 95%CI:0.800-1.381). No significant difference was observed in KK + KE genotype frequencies between the two groups (P = 0.863), with an odds ratio of 0.935 (95%CI:0.436-2.005). CONCLUSION: The K469E polymorphism of the ICAM-1 gene was not associated with the susceptibility of rheumatoid arthritis.

[Maternally inherited aminoglycoside-induced and nonsyndromic hearing loss in five Han Chinese pedigrees].

OBJECTIVE: To study the effect of the mitochondrial 12S rRNA mutations on aminoglycoside-induced and nonsyndromic hearing loss, to carry out the clinical and molecular characterization of five Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss. METHODS: Five pedigrees of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss were collected, genomic DNA was extracted, and complete mitochondrial genomes and the gap junction protein beta 2 (GJB2) gene were amplified and sequenced. RESULTS: Clinical evaluation revealed a wide range of severity, age-at-onset and audiometric configuration of hearing impairment in the matrilineal relatives in these families. The penetrance rates of hearing loss in these pedigrees were 17.6%, 50.0%, 66.7%, 31.3% and 23.1%, with an average of 37.7%, when aminoglycoside-induced deafness was included. Sequence analysis of the complete mitochondrial genomes in these pedigrees identified the known 1555A>G mutation and distinct sets of mitochondrial DNA(mtDNA) polymorphisms belonging to Eastern Asian haplogroups D4b2b, B4c1b1, F3, C1 and D5a, respectively. Of these variants, ND1 L89T and CO3 A200T mutations resided at the highly conservative regions. However, there were no functionally significant mutations in tRNAs and rRNAs or secondary known mutations. No hearing loss related GJB2 gene mutation was observed. CONCLUSION: The lack of significant mutation in the ruled out the possible involvement of GJB2 in the phenotypic expression of the 1555A>G mutation in those affected subjects. However, aminoglycosides, mtDNA variations and other nuclear modifier genes may play an important role in the phenotypic manifestation of the 1555A>G mutation in these Chinese families.

[Rapid and high-throughput multiplex ligation-dependent probe amplification for diagnosis of chromosome aneuploidy].

OBJECTIVE: To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice. METHODS: The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. RESULTS: The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples, the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of >1.3. CONCLUSION: Thedata suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.

[Association study on the mitochondrial genome region np16181-16193 variation with type 2 diabetes mellitus].

OBJECTIVE: To investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM). METHODS: Blood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms. RESULTS: The mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05). CONCLUSION: The mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.

Correlation between procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 and breast cancer.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), which affects collagen synthesis, is associated with breast cancer. The purpose of the study is to detect the expression of PLOD2 in breast cancer and to evaluate the correlation between PLOD2 and clinicopathologic characteristics and prognosis of patients with breast cancer. 50 paired samples including breast cancer tissues and adjacent non-tumor tissues were formalin-fixed and evaluated by immunohistochemistry. The results revealed that PLOD2 expression in breast cancer tissues was much higher than that in tissues adjacent to breast cancer. High expression of PLOD2 was positively associated with tumor stage (P = 0.003) and lymph node metastasis (P = 0.001). However, high expression of PLOD2 was negatively related to Ki-67 (P < 0.001) while positively related to progesterone receptor (PR) (P = 0.001). PLOD2 expression was positively related to the metastasis of breast cancer. Therefore, high expression of PLOD2 was identified as a poor prognostic biomarker for patients with breast cancer. These results suggest a novel molecular mechanism in breast cancer tumorigenesis, thus providing a potential therapeutic target of breast cancer.

[Hearing loss and epilepsy may be associated with the novel mitochondrial tRNASer(UCN) 7472delC mutation in a Chinese family].

Mutations in mitochondrial DNA have been associated with a wide spectrum of clinical abnormalities. We reported here the clinical and genetic evaluations as well as mutational analysis of mitochondrial DNA(mtDNA) in a three-generation Chinese Han family with maternally transmitted hearing loss and epilepsy. Of 14 matrilineal relatives, three suffered from hearing loss, three had epilepsy, and other did not have significant clinical abnormalities. Sequence analysis of mitochondrial genome in this family identified the novel 7472delC in tRNASer(UCN) and 33 variants belonging to Asian haplogroup B4b1a2. The 7472delC locates at the highly conserved residue of T-arm of this tRNA. In fact, the 7472insC at the same position of this tRNA has been associated with hearing loss and epilepsy in several genetically unrelated families. The 7472insC has been shown to lead to a failure in tRNA metabolism and mitochondrial dysfunction. Thus, 7472delC mutation, similar to 7472insC mutation, may result in the mitochondrial dysfunctions responsible for the hearing loss and epilepsy. Furthermore, none of mutation in deafness-associated GJB2 gene was detected in this Chinese family. Therefore, the 7472delC is likely a new mitochondrial mutation with hearing loss and epilepsy.

[Regulating effects of Paecilomyces cicadae polysaccharides on immunity of aged rats].

OBJECTIVE: To investigate the effects of Paecilomyces cicadae polysaccharide (PCPS) on the immunological function of aged rats in vivo. METHOD: The young and old rats were administered with normal saline as control groups, and the rats from test group were sc given 50, 100, 200 mg x kg(-1) x d(-1) dosage of PCPS for 3 weeks. The phagocytizing rate and index of PMphi, AMphi to S. aureas were observed, and the colorimetric MTI was used to analyze the proliferative activity of spleenocytes which had been stimulated with ConA or LPS. We also inspected the ability varing of ACP, LDH, ARG of spleen, and observed the ultramicro structure of spleen under the SEM. RESULT: The phagocytosis of Mphi was lower in aged group than that in young' s group, and the proliferative activity of spleenocytes was lower too. The activities of ACP, LDH, ARG of spleen were extremely decreased (P < 0.01) in aged rats as well. The proliferative activity and phagocytotic rate were both extremely increased in PCPS groups (P < 0.01), and the mitochondrion and endoplasmic reticulum of spleen were accrementition as well (P < 0.01). CONCLUSION: PCPS could enhance the phagocytizing function of PMphi, AMphi of aged rats in vivo, and strengthen the immune function of spleen and its proliferative activity as well. Then the immunity of aged rats could be improved. The PCPS may be an anti-aging agent.

[Leber's hereditary optic neuropathy is associated with the mitochondrial G11696A mutation in two Chinese families].

OBJECTIVE: To report the clinical, genetic, and molecular characterization of two Chinese families with Leber's hereditary optic neuropathy (LHON). METHODS: Ophthalmological examinations showed that only probands in two families exhibited visual loss at the age of 10 and 17 years respectively. The entire mitochondrial genome of two probands was PCR amplified in 24 overlapping fragments using sets of oligonucleotide primers. RESULTS: Mutational analysis of mitochondrial DNA (mtDNA) in these pedigrees revealed the absence of three common LHON associated G11778A, G3460A and T144484 mutations but the presence of homoplastic LHON associated ND4 G11696A mutation, which was present in one out of 167 Chinese healthy controls. CONCLUSION: Sequence analysis of the complete mitochondrial genomes in two pedigrees showed the distinct sets of mtDNA polymorphisms, belonging to Eastern Asian haplogroup D4. The incomplete penetrance of visual loss and the presence of one in 167 controls suggested that this mutation itself is insufficient to produce a clinical phenotype and other modifier factors play a role in the phenotypic manifestation. The lack of functional mtDNA variants in these pedigrees ruled out the role of mitochondrial background in the phenotypic expression of visual loss. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated G11696A mutation in two Chinese pedigrees.

Evaluation of a New IFN-γ Release Assay for Rapid Diagnosis of Active Tuberculosis in a High-Incidence Setting.

Blood-based interferon-gamma (IFN-γ) release assays (IGRAs) have been proven to be useful in the diagnosis of Mycobacterium tuberculosis (Mtb) infection. However, IGRAs have not been recommended for clinical practice in most low-income settings due to cost-intensive limitations and shortage of clinical data available. The established T-SPOT. TB assay containing Mtb-specific antigens ESAT-6 and CFP10 are widely used for immunodiagonsis of Mtb infection, but the high cost is one of the restricting factors against its clinical application in the developing countries. More recently, a cost-saving IGRA assay, TS-SPOT, was approved in China. This new assay contains an additional antigen Rv3615c. Rv3615c contains broadly recognized CD4+ and CD8+ epitopes, and T-cell responses to Rv3615c are as specific for Mtb infection as the responses to ESAT-6 and CFP10 in both Mtb-infected humans and M. bovis-infected cattle. Therefore, we assessed the likely effect of inclusion of Rv3615c as stimulus besides ESAT-6 and CFP10 in an IGRA assay and evaluated the performance of TS-SPOT for diagnosis of Mtb infection and active TB compared with T-SPOT.TB. We tested 155 active TB patients, 90 non-TB lung disease patients, and 55 healthy individuals. The results presented an improved positive rate for diagnosis of active TB and Mtb infection, that could be attributable to inclusion of Rv3615c in the mixture of stimulatory antigens. The diagnostic efficiency of TS-SPOT assay for active TB was as follows: sensitivity 80.00%, specificity 83.45%, positive predictive value (PPV) 83.78%, negative predictive value (NPV) 83.45%, positive likelihood ratio (LR+) 4.83, and negative likelihood ratio (LR-) 0.24. The results were similar to those of T-SPOT.TB, with an excellent agreement (κ = 0.91, 95% CI: 0.85-0.95) being observed between these two assays. The sensitivities of the TS-SPOT assay varied for patients with different forms of active TB, with the highest sensitivity for patients with culture-positive pulmonary TB (92.16%) and the lowest for those with tuberculosis meningitis (50.00%). Taken together, the current evidence indicates that this new TS-SPOT assay is a useful adjunct to the current tests for rapid diagnosis of active TB and Mtb infection in low-income and high-incidence settings due to its characteristics of cost-effectiveness and high-quality.

[Virological impact of stalk region of neuraminidase in influenza A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1) viruses].

This study aims to investigate the virological impact of the stalk region and cysteine (C) in neuraminidase (NA) of influenza A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1) viruses. The NA of A/ Anhui/1/05 (H5N1), defined as AH N1, lacked 20 amino acids (including C, defined as s20) as compared with NA of A/Ohio/07/2009 (H1N1) (defined as 09N1). We deleted s20 of 09N1 to construct 09N1-s20, and inserted s20 into AH N1 to construct AH N1+s20. To investigate the impact of C on the biological function of NA, we deleted C in 09N1 to construct 09N1-C and inserted C into AH N1 to construct AH N1-C. The pseudo-type viral particle (pp) system was used to evaluate the impact of these mutants on virology. The combination of 09N1-C and 09H1 (defined as 09H1::09N1-C) showed an infectivity 8 times that of the wild type 09H1::09N1, while the infectivity of the combination of AH N1+C and AH H5 (defined as AH H5::AH N1+C) was much lower than that of the wild type AH H5::AH N1. The infectivity of the combination of 09N1-s20 and 09H1 (defined as 09H1::09N1-s20) was 4 times that of the wild type 09H1::09N1; the infectivity of the combination of AH N1+s20 and AH H5 (defined as AH H5:: AH N1+s20) was 1/7 that of the wild type AH H5::AH N1. The co-existence of 09N1-C and AH H5 displayed 6 times the infectivity of AH H5::09N1, while the infectivity of 09H1::AH N1+C was very low. Multimer analysis showed that in the wild type 09N1, the forms of NA were dimer >> tetramer > monomer; the major component of NA in 09N1-C was monomer; in 09N1-s20, the forms of NA were monomer >> dimer. AH N1 was mainly composed of monomer; in AH N1+s20, the forms of NA were dimer >> monomer > tetramer; in AH N1+C, the forms of NA were dimer >> tetramer. Deletion of C or s20 from 09N1 did not change the expression of NA. The study suggested that deletion of C from the stalk region of NA in A/Ohio/07/2009 (H1N1) increases infectivity. Insertion of C into NA's stalk region of A/ Anhui/1/05 (H5N1) significantly decreases infectivity. Cysteine deletion in the stalk region is important for the infectivity of A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1). It may interfere with the infectivity via changes in NA polymerization.

[HIV-1 infection changes miRNA expression profile in the whole blood].

To describe the unique miRNA profiles for HIV seropositive individuals and identify significantly differently expressed miRNAs, we determined the expression level of 754 miRNAs of 10 HIV seropositive individuals and 10 HIV seronegative individuals by using the Taqman low density microRNA array. BRB-Array Tool was used to conduct the significance analysis, and the DIANA online tool was used to perform the miRNA target prediction and pathway analysis. A total of 56 significantly differentially expressed miRNAs were identified by microarray between HIV seropositive and seronegative individuals. Among them, 49 miRNAs were down-regulated and 7 were up-regulated, partially overlapped with reported data. Predicted target genes were mainly involved in MAPK, TGF-beta and Wnt pathways. The results shows that miRNA profile changes in HIV-1 seropositive individuals, and the 56 differentially expressed miRNAs may play important role during HIV infection. Further studies on these miRNAs may be helpful for identify key molecules involved in HIV infection and potential diagnostic markers.

[Development and identification of polyclonal antibodies against HIV-1 Vpr-derived polypeptides].

To develop polyclonal antibodies against predicted B cell epitopes in HIV-1 accessory protein Vpr, the prepared consensus Vpr amino acid sequence was used to predict potential B cell epitopes by online softwares (ABCpred and Bcepred), the synthesized polypeptides of B-cell epitopes were subsequently conjugated with keyhole limpet hemocyanin (KLH) and then used to immunize rabbits. The antibody titers were determined by ELISA, and antibody specifity was analyzed by Western-Blotting and immunoprecipitation, respectively. Amino acid residues 3-19 (N) and 82-95 (C) of Vpr were predicted as the potential B cell epitopes. After inoculation of the conjugation of synthesized peptide to KLH, the antibody titers in rabbit sera against N and C peptides reached more than 1:100000 by ELISA. Western-Blotting analysis showed that the polyclonal antibodies reacted with both wild Vpr and fusion protein of GFP with Vpr, no matter Vpr was derived from HIV-1 B subtype or CRF07_BC recombinant form; Immunoprecipitation analysis showed similar reactions to Western-Blotting results. Two B cell epitopes of Vpr were successfully predicted by Bio-informatics methods and polyclonal antibodies against those peptides could be successfully prepared.

[Detection of WU polyomavirus in children with low respiratory tract infections using real-time fluorescent quantitative PCR].

OBJECTIVE: Development and application of a real time fluorescent quantitative PCR (FQ-PCR) assay for detecting WU polyomavirus in children with low respiratory tract infections. METHODS: The VP2 gene of WU polyomavirus was selected as the detection target, from which the real time primers and probes were designed. The standard curve was established by using recombinant plasmid as template. And the FQ-PCR assay for specific detection of WU polyomavirus was established. The specificity, sensitivity and reproducibility of the method were evaluated. Furthermore, the clinical specimens from children with respiratory tract infections collected in Wenling First People's Hospital were quantitatively detected using this method. RESULTS: In this study, the FQ-PCR method was established to detect a specific fragment in VP2gene of WU polyomavirus. The standard curve coefficient R2 was 0.998. And this method can detect as low as 50 copies recombinant plasmid. The clinical specimens of sputum and throat swab from children with respiratory tract infections were quantitatively detected using this method. 7 sputum specimens were detected as WU polyomavirus positive in 700 sputum specimens, the positive ratio was 1.00%. No positive specimens were detected in 146 specimens of throat swabs and 846 blood samples from same patient population. CONCLUSION: The results indicated that the FQ-PCR assay method established in this study was specific, rapid and sensitive for detecting WU polyomavirus in children with lower respiratory tract infections. The sputum specimen is more suitable to be used for gene detection of WU polyomavirus than throat swab or blood.

[Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay].

OBJECTIVE: To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA). METHODS: The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA. RESULTS: CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability. CONCLUSION: The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.