Overall Evaluation and Management of 8 Instruments in Emergency Laboratory Using 6σ Quality Standard / 现代检验医学杂志

Objective To evaluate and improve overall quality detection of 8 instruments in emergency laboratory using 6σ quality standard.Methods In 2017,collected both internal quality control (IQC) and external quality assessment (EQA) data of 46 assay items from 8 instruments in their laboratory,and then calculated their σ values and total allowed errors ac cording to CLIA'88 standards.By comparison of their quality goal index (QGI) between first and second half year of 2017,successfully elevated assayquality using strategy (man,machine,material,method,measurment and enviroment).Results For all 46 assay items (except NT-proBNP without second EQA data),made better improvement of detection of 34 (75.6 ％ of 6σ qualified),38(84.4％ of 5σ qualified),40(88.9％ of 4σ qualified),44 (97.8％ of 3σ qualified) and 1 (2.2％ of 2～3σ qualified) items,respectively whereas there were 27 (58.7％ of 6σ qualified),30 (65.2％ of 5σ qualified),35 (76.1％ of 4σ qualified),41 (89.1％ of 3σ qualified) and 5 (10.9％ of 2～3σ qualified) items before improvement.Conclusion Using 6σ quality standard,could make more progress in the laboratory for many respects:fast and accurate clinical reports,reducing reinspection rate,and increasing patient's satisfactory,etc.

Mutant D178N prion protein converts spontaneously in RT-QuIC assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the conversion of mutant D178N prion protein in RT-QuIC assay.</p><p><b>METHODS</b>The D178N mutant prion PRNP was generated by the method of single site mutation. The mutant PRNP gene was inserted into plasmids of pET24. The full and N-truncated recombinant human prion proteins were expressed and purified. The fibril formations of these proteins were real-time monitored by the method of RT-QuIC. The ability to resist proteinase K (PK) of these fibrils was analyzed.</p><p><b>RESULTS</b>We succeed to construct human PrP-D178N plamids. The N-truncated human prion protein with D178N (PrP90-231-D178N) can convert spontaneously in RT-QuIC, while full length of human prion D178N protein (PrP23-231-D178N) fails to convert spontaneously. The spontaneously generated fibril has been domenstrated it is partily PK-resistant.</p><p><b>CONCLUSION</b>The N-terminal of prion protein (23-90) plays an important role for the D178N mutant protein spontaneously conversion, which provide the clues for study the pathogenesis of genetic CJD.</p>

Establishment of hamster- and human-PRNP transgenic mice / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs).</p><p><b>METHODS</b>Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods.</p><p><b>RESULTS</b>Integrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs.</p><p><b>CONCLUSION</b>We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.</p>

Mutations in various functional domains of HPV2 E2 protein inhibit the transcriptional depression activities / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the potential transcriptional depression activities of HPV2 E2 proteins with mutations in different functional domains.</p><p><b>METHODS</b>The primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptional repression activities of the E2 mutants were evaluated by detection of CAT expression values.</p><p><b>RESULTS</b>Compared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptional repressive activities. The point mutations in the transactivation domain (nt 3037), the internal hinge region (nt 3387) and DNA binding domain (nt 3697) showed remarkable inhibition on its transcriptional depression function.</p><p><b>CONCLUSION</b>The transcriptional regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.</p>

Molecular epidemiological study on prevalence of human papillomaviruses in patients with common warts in Beijing area / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the circulation, distribution, and genomic diversity of HPVs in common warts in Beijing area of China.</p><p><b>METHODS</b>Forty eight patients with pathologically diagnosed common warts were screened for the presence of HPV with HPV type-specific PCR and direct sequencing analysis. The genomic diversity of HPVs prevalent in Chinese patients was analyzed based on LCR.</p><p><b>RESULTS</b>Forty one (85.5%) samples were positive for HPV DNA, 13 (31.7%)--HPV-57, 12 (29.3%)--HPV-1a, 7 (17%)--HPV-27 and 5(12.2%)--HPV-2a. Four cases were infected with two different HPV types, two (4.9%) with HPV-1a and HPV-27, one (2.4%) with HPV-1 and HPV-57 and one (2.4%) with HPV-27 and HPV-57. In contrast to the prevalence of single strain of novel HPV-57 variant and HPV-1 prototype, two HPV-2 and three HPV-27 novel variants were found to circulate in Beijing.</p><p><b>CONCLUSION</b>HPV-1, -2, -27 and -57 are predominantly prevalent in patients with common warts in Beijing.</p>

Study on the phenomenon of splashes and sprays from virology / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the phenomenon of accidental splashes and sprays from manipulation of recombinant virus material and to measure the approximate spilled distance when recombinant virus material inadvertently dropped in the biosafety laboratory.</p><p><b>METHODS</b>first, two groups owning different experience simulated the course of accidental spills and splashes by recombinant adenovirus (rADV) which expressed green fluorescence protein (GFP), the GFP signal were observed in 96 well cell plate after spills appeared; Second, the routine two heights (75 cm and 110 cm) and capacity (1 ml, 1.5 ml, 4 ml and 8 ml) of virus were chose to simulate the experiment of unexpected dropping.</p><p><b>RESULTS</b>First, the positive quantity of the first group owning 5 years' experience is much less than the second group owning 2 years' work experience, the former was 7 positive wells, the latter was 81 positive when they used the pipette to operation. Second, when the unclosed test tubes (1 ml, 1.5 ml, 4 ml and 8 ml recombinant virus) inadvertently dropped, the largest spill distance was 0.92 m, 1.57 m, 2.63 m and2.68 m respectively.</p><p><b>CONCLUSION</b>The better experience is important to make sure safety when we make infectious material; the contaminated distance increased with the amount of recombinant virus material.</p>

Establishment of a stable PrP(Sc) panel from brain tissues of experimental hamsters with scrapie strain 263K / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.</p><p><b>METHODS</b>Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.</p>

Transcriptional repressive activity of mutated E2 protein of human papillomavirus 2 (HPV-2) variant / 病毒学报

Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart.

Expression of cytosolic PrP and analysis of its cytotoxic activities / 病毒学报

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.

The construction and identification of the PRNP vectors with ubiquitin or the lysosome-targeting signal / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate PrP expression characteristic of PRNP nucleic acid vaccine vector with ubiquitin or the lysosome-targeting signal.</p><p><b>METHODS</b>The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector that is, pcDNA3.1-UPrP and pcDNA3.1-PrPL were constructed. The expression characteristics of PrP with two signals were evaluated by Western Blot and the localization was observed by indirect immune fluorescence.</p><p><b>RESULTS</b>The protein expressed by pcDNA3.1-UPrP and pcDNA3.1-PrPL with ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP.PrP with ubiquitin was degraded gradually with time extension,whereas quantity of PrP with lysosome signal reduced in 48 h after transfection. The protein with two location signals can direct fusion proteins to cytoplasm.</p><p><b>CONCLUSION</b>The PRNP vectors with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. There will be one of good foundation on PRNP nucleic acid vaccine.</p>

Establishment of a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters infected with scrapie agent 263K / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.</p><p><b>METHODS</b>30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.</p>

Investigating the risk of two model virus in the laboratory / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the survival time of recombination rival in environment and inactivation ability of different disinfectant and ultraviolet radiation against virus.</p><p><b>METHODS</b>NC membranes absorbed the recombinant adenovirus (rADV) or herpes simplex virus (rHSV) with green fluorescence protein (GFP) were laid, or immersed in various concentration of different disinfectants such as ethanol, sodium hypochlorite, lysol and geramine and then taked out them every 15 min, or exposed under ultraviolet radiation, then the NC membranes were adsorbed 1 h in cell, 37 degrees C 5% CO2 48 h. The results were observed under the fluorescence microscope.</p><p><b>RESULTS</b>(1) the average survival time of rHSV under environment is less than 60 min, rADV is almost up to 2 h. (2) The infection ability of rHSV and rADV was inactived 15 min by both ethanol (100%, 70% and 50%) and sodium hypochlorite (5%, 2.5% and 1.25%). (3) Two virus can be killed by 0.1% bromogeramine. (4) Both 5% and 2.5% lysol, but rADV can not lost the infection on Vero Cell until 75 min by 1.25% Lysol. (5) The rHSV was inactivated under ultraviolet radiation, but rADV was not.</p><p><b>CONCLUSION</b>The survival time of is different from both envelope rival and the no-envelope viral under nature environment and the inactivate ability of disinfectant also is different between two model virus; Disinfectant should be choose according to virus type.</p>

Preliminary analyses for influence of mutant PrPs with different number of octapeptide / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.</p><p><b>METHODS AND RESULTS</b>Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.</p><p><b>CONCLUSION</b>These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.</p>

Generation and identification of phage engineering antibodies library against hamster prion protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein.</p><p><b>METHODS</b>Fab antibodies were identified and confirmed. BALB/c mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragments and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagemid vector pComb3.</p><p><b>RESULTS</b>The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10(6). The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed.</p><p><b>CONCLUSION</b>The research in this article will provide foundation for study of diagnosis and therapy of prion.</p>

Expression of recombinant human doppel protein and analysis of its cytotoxic activities / 病毒学报

Doppel (Dpl) is a newly identified PrP-associated protein. In this study, specific primers for human PRND gene were designed based on the human PRND cDNA sequence encoding human Dpl protein reported in the GenBank. The full-length PRND gene sequence with 531 bp long was obtained from DNA of human peripheral leucocytes as the template by polymerase chain reaction (PCR). After verified by sequence analysis, the PCR product was inserted into a prokaryotic-expressing vector and then transformed into E. coli JM109. The recombinant human Doppel protein (rhDpl) was expressed as inclusion bodies after IPTG induction, with the yield of more than 60% of total bacterial proteins. The rhDpl protein was purified by Ni2+ affinity chromatography and cleaved by hydroxylamine. SDS-PAGE revealed the molecular weight of the purified rhDpl protein was about 15 kD. Trypan blue and MTT assays identified that rhDpl in vitro inhibited the growth of human neuroblastoma cell line SH-SY5Y at concentrations > or =50 microg/mL and human cervical cancer cell line HeLa at concentrations > or =100 microg/mL, showing remarkably dose-and time-dependant manners. Hoechst33342-staining of SH-SY5Y cells treated with rhDpl showed massive apoptosis under fluorescent microscope. These results indicate that Dpl protein possesses cytotoxic activity in vitro, with obvious tissue-specific characteristics. This study provides the foundation for further study of Dpl biological functions in vitro and in vivo.

Protein expression of human neuron-specific enolase and its antiserum preparation / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.</p><p><b>METHODS</b>HuNSE gene was amplified by RT-PCR and subcloned into a HIS-tagged expression vector pQE30 after sequence verification. HIS-NSE fusion protein expression was obtained in E. coli M15 after IPTG induction followed by purification of the fusion protein by Ni-NTA affinity chromatography. Two male rabbits were immunized for 4 times with the purified protein, and the antiserum against NSE protein was collected and evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemistry.</p><p><b>RESULTS</b>SDS-PAGE assay yielded an approximately 22 kD HIS-NSE fusion protein. The prepared antiserum could recognize both recombinant NSE protein and native NSE protein extracted from the brain tissues of different mammalian species as shown by Western blotting and immunohistochemistry.</p><p><b>CONCLUSION</b>High expression of HuNSE is obtained in E. coli and the prepared antiserum against HuNSE can be used potentially for diagnosis of prion-associated diseases and other nervous degeneration diseases.</p>

Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases.</p><p><b>METHODS</b>Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry.</p><p><b>RESULTS</b>The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc.</p><p><b>CONCLUSION</b>The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.</p>

Establishment of a sandwich ELISA method for detection of matrix metalloproteinase 9 protein in sera samples and preliminary application for hepatocellular carcinoma patients / 中华检验医学杂志

Objective Expression of recombinant human matrix metalloproteinase 9 (MMP9) protein in E coli,To establish a sandwich ELISA method for detecting MMP9 in sera and analyses of the difference of serum MMP9 in health population and the patient with hepatocellular carcinoma (HCC). Methods A fragment MMP9 sequence from nt 721-1 156 was amplified with PCR from the synthesized cDNAs of human liver tissues and inserted into the prokaryotic-expression plasmid pQE30.HIS-MMP9 fusion protein was expressed and purified from E coli MIS cells.The specific antibodies were elicited in rabbits and guinea pigs by immunization of the purified HIS-MMP9.After purifing antibodies with CNBr-activated Sepharose 4B,a sandwich ELISA technique was established.The serum MMP9 proteins were evaluated in 227 health adults and 193 HCC patients.Results SDS-PAGE displayed that the molecular-weight of the expressed fusion protein was about 17 000.The prepared antisera were able to recognize both recombinant and endogenously expressed MMP9 from human neutrophil and HepG2 cells.It was found that the average level of MMP9 proteins in HCC patients was higher than that in health control,showing significantly statistic difference.Conclusions The established method could reflect the level of serum MMPg.The data in this study supply scientific basis of generating methodology for screening metastasis and reoccurrence of HCC, using serum MMP9 as index.