Construction of Predictive Model in 9 037 Patients with Stroke / 中国实验方剂学杂志

ObjectiveTo develop and validate a predictive model to individually predict the risk of patients with stroke in the eICU Collaborative Research Database for early clinical identification and intervention. MethodIndividual patient data （200 859 cases） from a national multicenter cohort study （eICU database） were selected， and the patients with stroke in neurological diseases （9 037 cases） were selected for statistical analysis. The main outcome was hospital mortality. The Glasgow Coma scale （GCS） was used to divide all patients with stroke into stroke in meridian and stroke in viscera （GCS≤14 for stroke in viscera and GCS=15 for stroke in meridian）. The patients were then divided into a training set and a test set according to 7∶3， respectively， to evaluate the differences in hospital mortality between the two groups. The multivariate logistic regression was used to analyze the related factors affecting the prognosis of the two groups， and a predictive model was established. Receiver operator characteristic （ROC） curves were used to assess the discrimination of the predictive model. ResultThe predictive model based on 9 037 patients with stroke was established. The predictors of the stroke in meridian （4 475 cases） included pulmonary infection， mechanical ventilation， acute physiology， and chronic health status scoring system Ⅳ （APACHE Ⅳ） score. The predictors of the stroke in viscera （4 562 cases） included anticoagulation therapy （AT）， mechanical ventilation， acute physiology， and APACHE Ⅳ score. According to the predictors， the predictive models of the stroke in meridian and the stroke in viscera were constructed， respectively. The areas under the curve （AUC） of ROC of the training set and the test set of the predictive models of the stroke in meridian were 0.845 ［95% confidence interval （CI） （0.811， 0.879）］ and 0.807 ［95% CI （0.751， 0.863）］， respectively. The areas under the ROC curve of the training set and test set of the predictive models of the stroke in viscera were 0.799 ［95% CI （0.781， 0.817）］ and 0.805 ［95% CI （0.778， 0.832）］， respectively. The AUC of the predictive model of the training set and the test set were both above 0.7. ConclusionThe model established in this study can conveniently， directly， and accurately predict the hospital mortality risk of patients with stroke. Physicians and other healthcare professionals can use this predictive approach to provide early care planning and clinical interventions for patients with stroke during their hospital stay.

Clinical and genetic analysis of a case with Thiamine metabolism dysfunction syndrome 5 / 中华医学遗传学杂志

OBJECTIVE@#To report the clinical manifestation and genetic characteristics of a child with Thiamine metabolism dysfunction syndrome 5.@*METHODS@#Clinical data and genetic results were collected and analyzed. Peripheral blood samples of the child and their parents were collected for whole exome sequencing, and the functional effect of the variants on the TPK1 enzyme activity was verified by an in vitro assay.@*RESULTS@#A four-year-old boy presented with preschool onset of ataxia were characterized. High-throughput sequencing identified a novel homozygous variant of TPK1 gene c.382G>A (p.Leu128Phe). His father and mother were both found carrying the variant. The variant protein showed a 30.9% reduction in TPK1 enzyme activity compared with the wildtype.@*CONCLUSION@#A novel pathogenic variant has been identified in a boy with thiamine metabolic dysfunction syndrome type 5.

Progress in HIV-1 Env trimer design / 生物工程学报

Currently, HIV-1 vaccine development has still been a hot pot in the AIDS research. HIV-1 glycoprotein Env is the sole target in the virion surface that mediates the membrane fusion between the virion and cell in the HIV-1 infection process. Env protein is the significant immunogen for HIV-1 vaccine development. In recent years, there have been breakthroughs in the Env trimer research. For example, the strategies including SOSIP, NFL2P, and UFO had been applied to design and generate HIV-1 Env trimer. The improvement of quantity and stability is beneficial to achieve the HIV-1 native-like Env trimer for elicitation of strong neutralizing antibody responsing in animal immunization. This review focuses on the different strategies for Env trimer design and compares their advantages and disadvantages, combining with our work to give some advice, which might provide relevant information for the future HIV-1 immunogen design.

Progress in vaccine development based on baculovirus expression vector system / 生物工程学报

Baculovirus expression vector system (BEVS) has been successfully applied to the over-expression of various proteins, thus providing sufficient materials for vaccine research. Compared to other systems, BEVS has many advantages: baculovirus solely being parasitic in invertebrates, the resultant products conferring high safety to mammalian, high expression level of recombinant proteins, preferable folding for eukaryotic protein, proper post-translational modification required for biological function, suitable for multiple genes co-expression and large-scale production with serum-free culture media. To better understand the advantages and prospective of BEVS for the vaccine research, this article will review the development of BEVS and its application on vaccine research.

Expression and self-assembly of HIV-1 CAP2NC protein / 生物工程学报

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay / 中华预防医学杂志

Objective@#To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.@*Methods@#HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.@*Results@#The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05).@*Conclusion@#We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Analysis of the serological testing results from Aier Eye Bank / 中华实验眼科杂志

Objective This study was to evaluate the safety of 640 corneal donors by analysing the serological testing results.Methods We retrospectively analyzed the serological testing results from Changsha Aier Eye Bank and Chengdu Kangqiao Aier Eye Bank from January 2011 to December 2015,hepatitis B virus surface antigen (HBsAg),hepatitis C virus (HCV),treponema pallidum (TP) and human immunodeficiency virus (HIV) were detected by colloidal gold or enzyme-linked immunosorbent assay (ELISA).Results There were 83 out of 640 serum samples showed positive immuno-reaction assayed markers,the positive rate was 12.97％,including HBsAg(n=60,9.38％),HCV(n=3,0.47％),TP(n=11,1.72％) and HIV(n=2,0.31％).Moreover,3 corneal donors were both positive against HBsAg and HCV,2 donors positive against HCV and TP,1 donor positive against HBsAg and HIV,1 donor positive against HBsAg and TP.Conclusions There is a high proportion of positive results of blood-borne diseases in cornea donors,which is a potential threat to corneal receptors and eye bank workers.Therefore,it is very important to detect serological test strictly for corneal donors.

Monoclonal antibodies against HPV11 virus-like particles：functional characteristics and application on quality assessment / 中国生化药物杂志

Objective To quantitatively analyze the characteristics of a panel of murine anti-human papillomavirus(HPV)11 L1-derived virus-like particle( VLP ) monoclonal antibodies ( mAbs ) and establish the mAb-based methods for antigen quality analysis.Methods A panel of 22 murine anti-HPV11 mAbs were characterized in details with their isotype, and binding affinity, conformational sensitivity were examined quantitatively in the direct binding ELISA and Western blot.The hemagglutination inhibition activity of mAbs were identified using the hemagglutination inhibition assay and the pseudovirus ( PsV ) neutralization efficiency were examined quantitatively using the PsV-based neutralization assay.The type-specific, highly conformational sensitive and neutralizing mAbs were selected to be used in the sandwich ELISA assay.Results Based on the quantitative and semi-quantitative results, six type-specific, highly conformational sensitive and neutralizing mAbs (2A2, 4A1-3, 16G7, 14A6, 9C1 and 19C7) were identified.These mAbs, along with 10D6 were screened as the capture mAb or as the detection mAb in the sandwich ELISA.Conclusion The binding affinity, conformational sensitivity and neutralization efficiency of anti-HPV11 mAbs were characterized in details.A mAb-based sandwich ELISA assay (14A6:Ag:9C12-HRP) were developed, which could be used in the in vitro potency analysis of HPV11 VLP-based vaccine.

Review for progress and current status of human papillomavirus prophylactic vaccine / 中国生化药物杂志

Persistant infection of high-risk human papillomavirus (HPV) is the primary cause leading to cervical cancer, which is ranked as second cancer threatening the health of women following breast cancer.Development of HPV vaccine is very important because there is no effective therapeutics for cervical cancer.Three currently licensed HPV vaccines based on major capsid protein L1 in the foreign market confered good safety and efficacy in clinical trials, but the current price is expensive due to high cost, which limits the wide application in developing countries.So far, the vaccines have not been launced in China market.Here, we review the progress and the current status of the HPV vaccine, which will attract the readers’ interest on the forthcoming emergence of HPV vaccine in China.

Establishment of human papillomavirus pseudovirion infection model in mouse for potency evaluation of HPV16 VLP Vaccine / 中国生化药物杂志

Objective To establish a mouse model of genital human papillomavirus (HPV) pseudovirion (PsV) transmission and evaluate the protective potency of HPV16 VLP vaccine.Methods HPV16 PsV with the encapsidated luciferase expressing plasmid Luc were generated from 293FT cells and purified by size-exclusion chromatography.The endpoint titers of HPV16 PsV-Luc were determined on 293FT cells, denoted as TRLU/mL.For in vivo genital challenge, mice were synchronized in a diestrus-like status by a subcutaneous injection with 0.1 μg β-estradiol and then with 3mg DepoProvera after 24 hours.Six hours prior to HPV16 PsV-Luc challenge, deeply anesthetized mice were intravaginally pretreated with 50 μL of 4%nonoxynol-9 ( N-9 ).HPV16 PsV-Luc was thoroughly mixed with 20 μL solution containing 4%carboxymethylcellulose ( CMC ) and intravaginally instilled using a positive-displacement pipette.Forty-eight hours after PsV-Luc challenge, mice were anesthetized and D luciferin was intravaginally instilled.After 3 minites, bioluminescence was measured with a cryogenically cooled Xenogen IVIS camera system.Then,the murine genital challenge model was used to determine the potency that HPV16 VLP vaccine is efficient at preventing HPV infection.Results HPV16 PsV-Luc was generated and purified from 293FT cells.HPV16 PsV-Luc was verified to containe L1 and L2 protein by Western blot.HPV 16 PsV-Luc successfully infected vaginal epithelial cells of mouse and the murine genital challenge model was established.To achieve consistent bioluminescence, the minimal dose of HPV16 PsV-Luc was 1.7 ×104 TRLU.The protective potency of HPV16 VLP vaccine was shown using this murine model.Our data showed that immune serum with over neutralizing antibody titer of 256-fold was sufficient to confer protection against HPV PsV genital infection .Conclusion The murine genital challenge model of HPV16 was successfully established, and the model is used to evaluate the potency of HPV16 VLP vaccine against in vivo genital HPV16 PsV challenge.Our model will benefit for the investigation of HPV neutralization and the potency evaluation of the HPV vaccine .

Preparation of monoclonal antibodies against neutrophil gelatinase-associated lipocalin (NGAL) and development of an antibody-based chemiluminescence immune quantification assay / 中国生化药物杂志

Objective To obtain monoclonal antibodies ( mAbs ) against neutrophil gelatinase-associated lipocalin ( NGAL ) and a chemiluminescense immune quantification assay based one paired mAbs.Methods Six-to-eight weeks old female BALB/c mice were immunized with the purified recombinant human NGAL antigen( rhNGAL) that was produced by the Escherichia coil expression system.The spleen was fused with hybridoma for screening anti-NGAL monoclonal antibodies by indirect ELISA.Western blot was implemented to identify the reactivity with native NGAL. Results The rhNGAL antigen was found to form disulfide cross-linked dimers and present excellent immunogenicity.The reaction titer of the immune serum of NGAL immunized mice was about 106.Thirty mAbs were screened by indirect ELISA, hereinto;the EC50 values of mAb23C12 and 38D10 were 0.034 g/mL, 0.022 g/mL respectively.The antibodies pair, 38D10/23C12-SAE labeled with AcridiniumEster(AE), were shown to work well in chemiluminescense immune response quantitative detection which was screened by NGAL standardand clinical urine samples.This detection can resolve positive and negative samples with a statistically significant difference (P<0.0001).And the correlation coefficient R2between NGAL quantitative results and that of the Abbott's NGAL chemiluminescence immune assay kit was greater than 0.97.The detection linear range was 10-1500 ng/mL, analytical sensitivity of the method was 0.63 ng/mL.Conclusion Highly purified rhNGAL antigen and specific anti-NGAL monoclonal antibodies are generated in this study.The detection capability of method is comparable with that of the international commercial kit.

Comparison of early developmental differences of hair follicles in different skin areas of neonatal mice / 中国实验动物学报

Objective The aim of this study was to observe the growth difference and expression of cytochrome C of skin hair follicles in neonatal mice .Methods The morphology of different skin hair follicles of neonatal mice ( postnatal day 1-9)were observed by HE staining histology and cytochrome C was detected by immunohistochemistry .Results The skin hair follicles in different parts of neonatal mice showed differences not only in morphology but also in developmental pe -riods.Hair follicle growth in the back and tail skin had a nonlinear and growing period .After the nonlinear and growing pe-riod they began to grow rapidly .The tail development was slightly slower than that on the back .The hair follicles of vibris-sae were very special , and started to develop without a stable period .Conclusions The results of morphological observa-tion and cytochrome C immunohistochemistry demonstrate that differences exist in the hair follicle morphology and develop -mental times in the skin of different parts of the body in neonatal mice .

Study on genetic transformation of antigen G3VP7 gene of human rotavirus in peanut / 生物医学工程学杂志

In order to increase the expression level of target gene and to simplify the purifying process of separation and purification, we performed the transgenetic research of antigen VP7 gene into peanut via Agrobacterium tumefaciens. The plant binary expression vector is pBOG3VP7 harboring fusion gene oleosin-vp7, which is promoted by ole-promoter. Cotyledon nodes were used as transformation recipients. Transformed individuals were obtained through selection on medium containing 125 mg L-1 Kan. Integration of transgenes was assessed by PCR amplification and PCR-Southern blot hybridization. Taking pBOG3VP7 plasmid as positive control, non-transformed peanut as negative control. 6 plants among 11 plants grown up through seletion medium were detected by PCR and the rate of positive plants is 54.5%. PCR positive plants were further analysed by PCR-Southern blot hybridization. The results showed that 3 plants have DNA bloting bands. The results also showed that the foreign gene was integrated into genome of transformed peanuts. Elevated expression of rotavirus VP7 antigen in transgenic peanuts was a critical factor in the development of efficient and cheap plant oral vaccine.

A novel potential neutralizing epitope of hepatitis E virus / 中华微生物学和免疫学杂志

Objective To search for the neutralizing epitopos on hepatitis E virus (HEV) capsid besides the known neutralizing epitope (aa459-606). Methods By analysis of several strains of monoclonal antibodies against HEV capsid and their recognized epitopes, the neutralizing activity of epitope (aa394-458) at N-terminus was compared with that of an immunodominant neutralizing epitope (an459-606). Re-suits The research showed a novel potential neutralizing epitope in aa423-437 of HEV ORF2 though detec-ting and comparing the characteristics of several antibodies and corresponding determinations. The epitope is a linear non-immunodominant epitope which is different from the other neutralizing epitope in aa459-606.And the amino acids sequence of this novel epitope is conservative. Conclusion ORF2 aa423-437 is a no-vel potential neutralizing leaner epitope of HEV. It is believed that the present work adds fundamental knowl-edge to our understanding of HEV capsid domain and contributes to the prevention and control of this dis-ease.

Expression of H5N1 avian influenza virus haemagglutinin protein in pichia pastoris by high-density cell fermentation / 生物工程学报

We produced high pathogenic avian influenza H5N1 haemagglutinin protein HA1 in recombinant Pichia pastoris in a 10 L fermentor, to establish a high-density cell fermentation method. We studied the effects of different factors such as culture temperature, induced temperature, methanol feeding methods, trace elements on the growth of Pichia pastoris, the yield and the biologic activity of recombinant HA1 protein. The culture temperature in pre-induced and induced stage were optimized at 25 degrees C to adapt cell growth and recombinant protein expression, and induced temperature at 25 degrees C also resulted in higher biologic activity of rHA1 than at 30 degrees C. The binding activity of rHA1 against a wide-spectrum neutralizing antibody was susceptible to the presence of any trace elements, although trace elements would essentially benefit for the cell fermentation. As a conclusion, the expression level of rHA1 produced with optimized fermentation process reached 120 mg/L, which was 10.5 times higher than the one produced in regular shaking flask. The resultant high-density cell fermentation can likely produce rHA1 of H5N1 in large scale.

Production of human papillomavirus type 6 virus-like particles and detection of its neutralizing antibodies / 中华微生物学和免疫学杂志

Objective To produce human papillomavirus type 6(HPV-6)virus-like particles with Escherichia coli expression system and study its immunogenicity.Methods HPV-6 L1 gene was inserted into pmkaryotic expression vector pTO-T7 and then expressed in Escherichia coli ER2566.The HPV-6 L1 protein was purified by ammonium sulfate precipitation,ion-exchange chromatography,and hydrophobic interaction chromatography.Then the purified HPV-6 L1 self-assembled into virus-like particle after removing 1,4dithiothreitol(DTr).The morphology of the virus-like particles was investigated with dynamic light scatter and transmission electron microscopy,and the immunogenicity was determined with in vitro pseudownons neutralization as8ay.Results HPV-6 L1 was expressed in soluble form in Escherichia coli.Following the removal of DTT,purified HPV-6 L1 protein could assemble into virus-like particles as 25 am in the radius.And the animal immunization test showed HPV-6 virus-like particles can elite hish titer neutralizing antibodies.Conclusion The bacterially expressed HPV-6 L1 VLP is highly immunogenieity and easy to produce.And it can be good candidate of HPV-6 vaccine.

Expression, purification and immunogenicity analysis of HPV type 18 virus-like particles from Escherichia coli / 生物工程学报

Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.

Conjunctival resection combined with amniotic membrane transplantation for recurrent immunologic disorders of cornea and conjunctiva / 眼科

Objective To report the clinical results of the treatment for recurrent immunologic disorders of cornea and conjunctiva by conjunctival resection with amniotic membrane transplantation(AMT). Design A retrospective clinical case series. Participants Five patients 5 eyes with recurrent immunologic disorders of cornea and conjunctiva were enrolled into this study, including 2 eyes of phlytenular keratoconjunctivitis with corneal lesion, 1 eye of nodular episcleritis, 2 eyes of corneoscleral interstitial keratitis. Methods Patients were treated with partial conjunctiva resection and AMT. After operation, steroids and cyclosporin eye-drops were given for 2-3 weeks, and patients were followed up for 6-12 months. Main Outcome Measures Complications and recurrent situations were observed. Results The corneal disease kept quiescent, and the blood vessel subsidized. There were no recurrences during the follow up period in 4 cases. Recurrent signs were noted in one patient at 12 months after surgery. But no recurrence of corneal lesion was noted although the conjunctivitis was severe. Conclusion Local conjunctiva resection combined with AMT is a useful method for recurrent cases of immunologic disorders of cornea and conjunctiva. (Ophthalmol CHN, 2006,15:177-179)