Survey on the Construction Status of Forensic Virtual Autopsy Laboratory and the Applicability of Laboratory Accreditation / 法医学杂志

OBJECTIVES@#To survey the development status and actual needs of virtual autopsy technology in China and to clarify the applicability of forensic virtual autopsy laboratory accreditation.@*METHODS@#The questionnaire was set up included three aspects：(1) the current status of virtual autopsy technology development; (2) the accreditation elements such as personnel, equipment, entrustment and acceptance, methods, environmental facilities; (3) the needs and suggestions of practicing institutions. A total of 130 forensic pathology institutions were surveyed by online participation through the Questionnaire Star platform.@*RESULTS@#Among the 130 institutions, 43.08% were familiar with the characteristics of virtual autopsy technology, 35.38% conducted or received training in virtual autopsy, and 70.77% have establishment needs (including maintenance). Relevant elements were suitable for laboratory accreditation.@*CONCLUSIONS@#Virtual autopsy identification has gained social recognition. There is a demand for accreditation of forensic virtual autopsy laboratory. After the preliminary assessment, considering the characteristics and current situation of this technology, China National Accreditation Service for Conformity Assessment (CNAS) can first carry out the accreditation pilot of virtual autopsy project at large comprehensive forensic institutions with higher identification capability, and then CNAS can popularize the accreditation in a wide range when the conditions are suitable.

Automatic Identification of Brain Injury Mechanism Based on Deep Learning / 法医学杂志

OBJECTIVES@#To apply the convolutional neural network (CNN) Inception_v3 model in automatic identification of acceleration and deceleration injury based on CT images of brain, and to explore the application prospect of deep learning technology in forensic brain injury mechanism inference.@*METHODS@#CT images from 190 cases with acceleration and deceleration brain injury were selected as the experimental group, and CT images from 130 normal brain cases were used as the control group. The above-mentioned 320 imaging data were divided into training validation dataset and testing dataset according to random sampling method. The model classification performance was evaluated by the accuracy rate, precision rate, recall rate, F1-value and AUC value.@*RESULTS@#In the training process and validation process, the accuracy rate of the model to classify acceleration injury, deceleration injury and normal brain was 99.00% and 87.21%, which met the requirements. The optimized model was used to test the data of the testing dataset, the result showed that the accuracy rate of the model in the test set was 87.18%, and the precision rate, recall rate, F1-score and AUC of the model to recognize acceleration injury were 84.38%, 90.00%, 87.10% and 0.98, respectively, to recognize deceleration injury were 86.67%, 72.22%, 78.79% and 0.92, respectively, to recognize normal brain were 88.57%, 89.86%, 89.21% and 0.93, respectively.@*CONCLUSIONS@#Inception_v3 model has potential application value in distinguishing acceleration and deceleration injury based on brain CT images, and is expected to become an auxiliary tool to infer the mechanism of head injury.

Logistic Regression Analysis of the Mechanism of Blunt Brain Injury Inference Based on CT Images / 法医学杂志

OBJECTIVES@#To study the correlation between CT imaging features of acceleration and deceleration brain injury and injury degree.@*METHODS@#A total of 299 cases with acceleration and deceleration brain injury were collected and divided into acceleration brain injury group and deceleration brain injury group according to the injury mechanism. Subarachnoid hemorrhage (SAH) and Glasgow coma scale (GCS), combined with skull fracture, epidural hematoma (EDH), subdural hematoma (SDH) and brain contusion on the same and opposite sides of the stress point were selected as the screening indexes. χ2 test was used for primary screening, and binary logistic regression analysis was used for secondary screening. The indexes with the strongest correlation in acceleration and deceleration injury mechanism were selected.@*RESULTS@#χ2 test showed that skull fracture and EDH on the same side of the stress point; EDH, SDH and brain contusion on the opposite of the stress point; SAH, GCS were correlated with acceleration and deceleration injury (P<0.05). According to binary logistic regression analysis, the odds ratio (OR) of EDH on the same side of the stress point was 2.697, the OR of brain contusion on the opposite of the stress point was 0.043 and the OR of GCS was 0.238, suggesting there was statistically significant (P<0.05).@*CONCLUSIONS@#EDH on the same side of the stress point, brain contusion on the opposite of the stress point and GCS can be used as key indicators to distinguish acceleration and deceleration injury mechanism. In addition, skull fracture on the same side of the stress point, EDH and SDH on the opposite of the stress point and SAH were relatively weak indicators in distinguishing acceleration and deceleration injury mechanism.

Evaluation and Countermeasures of the Implementation of Forensic Clinical Identification Standards Based on the Perspective of Accreditation / 法医学杂志

The new Standardization Law, implemented in 2018, has added a standard post-implementation evaluation system, aiming to continuously improve the quality of standards through post-implementation evaluation. Standards in the forensic science field are closely related to accreditation activities. Forensic science standards are not only the criteria on which accreditation activities are carried out, but also one of the key contents of the inspection of forensic science institutions in accreditation activities. Since 2018, the certification and accreditation policies in the forensic science field have also been changed, which has brought impacts on the construction of a standard system based on accreditation.This paper analyzes the standard data from China National Accreditation Center from Conformity Assessment on forensic clinical identification accreditation assessment. It points out that the current coverage of laboratory accreditation activities is limited, the development in different provinces is unbalanced, and there is overlap and crossover in the standards in use. It is emphasized that the construction of the national forensic science standardization technical committee, the improvement of the forensic science standard system, the establishment of the standard implementation evaluation index system, and promotion of the coordination of standards, and the certifications and accreditations should be accelerated, in order to continue to promote the standardization and accreditation activities in the field of forensic science.

Effect of BRMS1 expression on proliferation, migration and adhesion of mouse forestomach carcinoma

OBJECTIVE@#To discuss the effect of BRMS1 on the proliferation, migration and adhesion of mouse forestomach carcinoma.@*METHODS@#The constructed pCMV-myc-BRMS1 recombinant plasmid and blank plasmid were transfected into mouse forestomach carcinoma. MTT method was employed to measure the activity of gastric cancer cell; the scratch assay and Transwell assay to measure the migration and invasion of gastric cancer cell; the adhesion assay to measure the adhesion of gastric cancer cell; while the Western blot assay to measure the expression of The NF-κB signal pathway, downstream matrix metalloproteinase (MMP)-2, MMP-9 and osteopontin and E-cadherin in the gastric cancer cell. Besides, the transplanted animal model of gastric cancer in mice was constructed to measure the size of tumor xenograft.@*RESULTS@#Results of MTT assay showed that, compared with the empty vector control group, the activity of gastric cancer cell was not affected in the BRMS1 transfection group. The improved expression of BRMS1 could inhibit the adhesion, migration and invasion of gastric cancer cell (P < 0.01). Besides, compared with the empty vector control group, the phosphorylation of NF-κB p65 and IκBα was reduced in the BRMS1 transfection group, with the decreased expression of MMP-2, MMP-9 and osteopontin and the increased expression of E-cadherin (P < 0.01). Results of animal experiment also showed that the expression of BRMS1 did not affect the transplanted tumor.@*CONCLUSIONS@#The expression of BRMS1 can significantly inhibit the adhesion, migration, invasion and metastasis of mouse forestomach carcinoma gastric cancer cell, which is related to The NF-κB signal pathway.

Effect of BRMS1 expression on proliferation, migration and adhesion of mouse forestomach carcinoma

Objective: To discuss the effect of BRMS1 on the proliferation, migration and adhesion of mouse forestomach carcinoma. Methods: The constructed pCMV-myc- BRMS1 recombinant plasmid and blank plasmid were transfected into mouse forestomach carcinoma. MTT method was employed to measure the activity of gastric cancer cell; the scratch assay and Transwell assay to measure the migration and invasion of gastric cancer cell; the adhesion assay to measure the adhesion of gastric cancer cell; while the Western blot assay to measure the expression of The NF-κB signal pathway, downstream matrix metalloproteinase (MMP)-2, MMP-9 and osteopontin and E-cadherin in the gastric cancer cell. Besides, the transplanted animal model of gastric cancer in mice was constructed to measure the size of tumor xenograft. Results: Results of MTT assay showed that, compared with the empty vector control group, the activity of gastric cancer cell was not affected in the BRMS1 transfection group. The improved expression of BRMS1 could inhibit the adhesion, migration and invasion of gastric cancer cell (P < 0.01). Besides, compared with the empty vector control group, the phosphorylation of NF-κB p65 and IκBα was reduced in the BRMS1 transfection group, with the decreased expression of MMP-2, MMP-9 and osteopontin and the increased expression of E-cadherin (P < 0.01). Results of animal experiment also showed that the expression of BRMS1 did not affect the transplanted tumor. Conclusions: The expression of BRMS1 can significantly inhibit the adhesion, migration, invasion and metastasis of mouse forestomach carcinoma gastric cancer cell, which is related to The NF-κB signal pathway.

Detection technologies of microRNA and their prospects for forensic applications / 法医学杂志

MicroRNA (miRNA) belongs to a class of small, non-coding RNA molecules that contains 18-25 nucleotides and regulates gene expression at post-transcriptional level. Many miRNAs are highly conserved and display timing- and tissue-specific expression. With the advance of the miRNA detection technologies, miRNA has been introduced to forensic science as a potentially novel set of genetic markers of forensic body fluid identification, species identification and PMI estimation. In this article, the detection methodologies of miRNA are reviewed, and their potential applications in forensic practice and research future are also discussed.

Polymorphisms of 21 short tandem repeat loci of Salar minority ethnic group in Qinghai Province / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the polymorphisms of 21 short tandem repeat (STR)loci of Salar minority ethnic group in Qinghai Province.</p><p><b>METHODS</b>Blood samples were collected from 120 unrelated healthy Salar individuals from Gandu town in Hualong county. DNA templates were screened by home-made AGCU21+1 kit. The findings were further compared with those of Hans in Zhejiang Province, Hans in Ningxia Hui Autonomous Region, Tibetans in Tibet Autonomous Region, and Tujias in Hubei Province.</p><p><b>RESULTS</b>The allele frequencies of 21 STR loci ranged 0.0042-0.4917, the genotype frequencies ranged 0.0083-0.3750, the power of discrimination ranged 0.796-0.948, the heterozygosity ranged 0.650-0.817, the polymorphism information contents ranged 0.590-0.810, and the power of exclusion ranged 0.355-0.630. The cumulative coupling probability was 1.75×10(-20), and the cumulative power of exclusion was 0.9999999. Significant differences were found at 14, 12, 12, 13 of the 21 STR loci between Salar and Hans of Zhejiang Province, Ningxia Hui Autonomous Region, Tibetans of Tibet Autonomous Region, and Tujias of Hubei Province (P<0.05).</p><p><b>CONCLUSION</b>The 21 STR loci used in this study are highly polymorphic in Salar minority ethnic group from Qinghai Province and therefore suitable for population genetics study, screening of disease-related genes, and forensic individual identification.</p>

Surgical management and outcome of solid-pseudopapillary tumor of pancreas: a series of 58 cases / 中华外科杂志

<p><b>OBJECTIVE</b>To study the surgical management of solid-pseudopapillary tumor of the pancreas (SPTP) and its characteristics of outcome.</p><p><b>METHODS</b>Fifty-eight patients with SPTP of the pancreas admitted from January 2001 to December 2010 were retrospectively analyzed. There were 7 male and 51 female patients, with an average age of 30 years (ranging 9 to 70 years). Most patients were symptomatic before admission; the most common symptom was abdominal pain. Of the 58 patients, 21 patients underwent pancreaticoduodenectomy, 30 patients underwent distal pancreatectomy, 6 patients underwent central pancreatectomy, 1 patient underwent simple tumor enucleation, and 1 patients underwent duodenum-preserving pancreatic head resection.</p><p><b>RESULTS</b>The average length of stay in hospital was 23.8 days (ranging 12 to 64 days). Thirteen patients (22.4%) developed postoperative complications, including grade A postoperative pancreatic fistula of 8 cases, gastrointestinal tract bleeding of 1 case, pleural effusion of 2 cases, wound infection and fat liquefaction of 2 cases. Two patients underwent reoperation due to gastrointestinal tract bleeding or wound infection. There was no hospital death. Forty-four patients were followed-up for 7 to 136 months with an average of 41 months. All the 44 patients were alive, while 8 patients developed dyspepsia and 4 patients developed diabetes mellitus. There were no tumor recurrences or metastasis.</p><p><b>CONCLUSIONS</b>SPTP is found primarily in young women. Excellent prognosis would be achieved with surgical resection.</p>

Simultaneous liver-kidney transplantation in polycystic kidney and hepatic disease with kidney and liver failure / 中华外科杂志

<p><b>OBJECTIVE</b>To review the clinical experiences concerning simultaneous liver-kidney transplantation in polycystic kidney and hepatic disease with kidney and liver failure.</p><p><b>METHODS</b>This study involved 8 cases of simultaneous liver-kidney transplantation in polycystic kidney and hepatic disease with kidney and liver failure. There were 5 male and 3 female patients, aged from 41 to 67 years old with a mean of 52.8 years old. Six cases transplanted kidney after liver with orthotopic liver transplantation, and 2 cases transplanted liver after kidney with piggy-back liver transplantation. The acute rejections, complications, liver function, kidney functions, and survival rates of patient/liver/kidney were recorded.</p><p><b>RESULTS</b>Within the follow-up of 28 to 65 months, all 8 patients are still alive with normal liver and kidney functions: 2 living more than 5 years, 2 living more than 4 years and 4 living more than 2 years. 2 cases of pleural effusion and 1 case of pneumonia were complications after operation, which had been cured successfully. No acute rejection of allograft was observed.</p><p><b>CONCLUSIONS</b>Simultaneous liver-kidney transplantation is a safe and effective treatment for polycystic kidney and hepatic disease with kidney and liver failure.</p>

Factors responsible for inter-individual variations in dosage/concentration of tacrolimus in renal transplant recipients / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the factors responsible for the inter-individual variations in the dosage/concentration of tacrolimus in renal transplant recipients.</p><p><b>METHODS</b>This study involved renal transplant recipients receiving immunosuppressive therapy with the tacrolimus, mycophenolate and prednisone regimen after the operation. The gender, age, height, body weight, tacrolimus dosage, hormone dosage, diarrhea, blood lipids, liver function, renal function, albumin, and hematocrit of the patients were recorded at different time points, namely in early stage (3, 7, 14, and 30 days postoperatively, 118 cases), at 3 months (103 cases), 6 months (75 cases) and over one year (119 cases) after the operation. The concentrations of tacrolimus and gene polymorphisms at CYP3A5, MDR1 3435, MDR1 2677 and MDR1 1236 were also determined in these patients. Multiple linear regression was used for analysis of these factors with tacrolimus concentration/dosage*body surface area as the independent variable.</p><p><b>RESULTS</b>Patients in early stage following renal transplantation showed rather poor fitting of the stepwise regression model, which increased obviously 3 months after the operation and further increased till reaching a stable level at 6 months. Multiple factors were found to affect tacrolimus concentration/dosage in the early postoperative stage, during which period these factors underwent drastic variations and became stable 3 months later. In terms of pharmacogenomics, the major factors affecting tacrolimus concentration/dosage included MDR1 3435, MDR1 2677 and MDR1 1236 polymorphisms, which vastly varied between the patients early after the operation. Of these polymorphic sites, CYP3A5 produced only minor effects on tacrolimus concentration/dosage, and was not included as an active factor until the stable phase (over 1 year) following the transplantation; MDR1 3435 was found to be the predominant factor affecting tacrolimus metabolism in the stable phase. Age, liver function, albumin and hematocrit were found to be positively correlated to the independent variable tacrolimus concentration/dosage*body surface area, and identified as important factors responsible for the intra-individual variation of tacrolimus dosage/concentration.</p><p><b>CONCLUSION</b>The variations in the factors affecting tacrolimus dosage/concentration after renal transplantation are consistent with the clinical features of the patients, and these factors vary with the postoperative stages. Pharmacogenomic factors produce the most conspicuous effect on tacrolimus dosage/concentration, and agents that may interfere with tacrolimus metabolism should be avoided after the operation. Age, liver function, albumin and hematocrit are also important factors responsible for the variation of tacrolimus dosage/concentration.</p>

Expression of pS2, TGF-alpha and PCNA in the gastric mucosa of young rats with endotoxemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Growth, regeneration and reparation of gastric mucosal epithelium may relate to the expression of peptides. This study aimed to investigate the effect of pS2, TGF-alpha and PCNA in endotoxin-induced acute gastric mucosal injury in young rats.</p><p><b>METHODS</b>Eighteen-day-old Wistar rats were randomly injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg) or normal saline (control). The gastric mucosal specimens were harvested 1.5, 3, 6, 24, 48, and 72 hrs after LPS or normal saline injection (n=8 each). The pathological changes of the gastric mucosa were observed by hematoxylin-eosin staining. The expression of pS2,TGF-alpha and PCNA was measured by immunohistochemistry SP method.</p><p><b>RESULTS</b>Gastric mucosal injuries were the most serious 6 hrs after LPS injection, characterized by massive erosion, bleeding and cord necrosis of the gastric mucosa paralleling with gastric longitudinal axis. PCNA expression in the gastric mucosa in the LPS group 3, 6, 24 and 48 hrs after LPS injection was significantly lower than that in the control group (P<0.01). pS2 expression in the gastric mucosa weakened 1.5 hrs after LPS injection, recovered to the control level at 3 hrs and was significantly higher than the control at 6, 24, 48 and 72 hrs of LPS injection (P<0.01). TGF-alpha expression in the gastric mucosa in the LPS group increased significantly 6, 24 and 48 hrs after LPS injection when compared with the control group (P<0.01).</p><p><b>CONCLUSIONS</b>PCNA expression may be associated with the proliferation activity of the gastric mucosa in the process of gastric mucosal injury/reparation. pS2 and TGF-alpha might participate in the defense and reparation of gastric mucosal cells through mediating cell proliferation following acute gastric mucosal injury.</p>

Evaluation of the ultrasonographic features of axillary lymph node metastasis in breast cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To analyze the ultrasonographic features of axillary lymph node metastasis in breast cancer patients, and to evaluate the accuracy of these features by Receiver Operating Characteristic (ROC) curve.</p><p><b>METHODS</b>Ultrasonography was carried out in 113 patients (mean age 56.5 yr, range 29 - 77 yr). The ultrasonographic features of the primary tumor and the axillary lymph node were observed. Univariate analysis was performed by Chi-squared test and Fisher exact test, and multivariate analysis to determine independent significant individual variables by multiple logistic regression analysis. ROC curve analysis was done to determine the sensitivity and specificity of individual and combined ultrasonographic features in distinguishing metastatic lymph node from the normal one.</p><p><b>RESULTS</b>The primary tumor size, abundance of blood supply in the primary tumor, longitudinal-transverse diameter ratio, cortex-hilum thickness ratio (on the longitudinal section), abnormal cortex thickness, distribution of intra-nodal vascularity of axillary lymph nodes were found to be statistically significant factors by univariate analysis. The primary tumor size, longitudinal-transverse ratio and cortex-hilum thickness ratio of lymph node were proved to be significant independent predictors of axillary lymph node metastasis by logistic regression analysis. Through ROC analysis, the combination of these independent ultrasonographic feature predictors was found to contribute significantly in differentiating metastatic lymph node from the normal with a sensitivity of 88.6% and specificity of 84.5%.</p><p><b>CONCLUSION</b>Axillary ultrasonography is helpful in staging the axillary lymph node in breast cancer patient. The primary tumor size, longitudinal-transverse diameter ratio and cortex-hilum thickness ratio of lymph node are the main features to determine whether metastatic involvement is present or not.</p>

Immunogenicity of Different Genetic Type Rotavirus NSP4 in Mice / 中国生物工程杂志

NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i. m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELJSA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.

Radiofrequency ablation of recurrent hepatocellular carcinoma after hepatectomy / 中华外科杂志

<p><b>OBJECTIVE</b>To assess the efficiency and safety of radiofrequency ablation (RFA) of recurrent hepatocellular carcinoma (RHCC) after hepatectomy and to investigate efficacy of RFA for patients with early and late phase recurrence, separately, setting 1 year as the cut-off between the early and late phases.</p><p><b>METHODS</b>A total of 42 patients with 77 RHCC and a history of hepatic resection for hepatocellular carcinoma (HCC) underwent ultrasound-guided percutaneous radiofrequency ablation in our department and entered this study (RHCC group). The average diameter of RHCC was (3.8 +/- 1.4) cm (range, 1.5-6.6 cm). 21 of the 42 RHCC patients had Child-Pugh class A cirrhosis 19, class B and two, class C cirrhosis. The average interval between initial surgery and the diagnosis of recurrence was 22.8 months (range, 1-96 month). 42 RHCC patients were divided into two groups as early recurrence group including 20 patients with 40 RHCC, and late recurrence group including the other 22 patients with 37 RHCC according to the recurrence interval. During the same period 148 patients with 217 primary HCC were also treated by RF ablation and regarded as primary HCC group. The average diameter of primary HCC was (4.0 +/- 1.4) cm (range, 1.2-7.0 cm). Regular follow-up with enhanced CT was performed to evaluate the treatment results. Ablation was considered a success if no contrast enhancement was detected in the treated area on 1 month CT scans.</p><p><b>RESULTS</b>The ablation success rate, local recurrence rate, new tumor incidence and mean survival in RHCC group were 90.5%, 14.3%, 38.1% and (28.0 +/- 3.5) months, respectively, which were similar to the corresponding results of 87.2%, 16.2%, 37.8% and (39.0 +/- 2.1) month in primary HCC group. However, when further comparison was performed between early recurrence group, late recurrence group and primary HCC group, there were some significant differences. The incidence of new tumors in early recurrence group was significantly higher than that in late recurrent group (60.0% vs. 18.2%, P = 0.005); early recurrence group survived shorter than primary HCC group [(15.4 +/- 2.3) vs. (39.0 +/- 2.1) months, P < 0.005]. The survival time was similar between late recurrence group and primary HCC group. One case was found haemorrhage after RFA and recovered with conservative treatment. No major complications occurred in the remaining 41 patients.</p><p><b>CONCLUSIONS</b>RF ablation is generally effective and safe in treating RHCC. And it's more effective in late recurrence than in early recurrence.</p>

Study on the RNA of severe acute respiratory syndrome (SARS) associated coronavirus in the blood and excretion of convalescent patients with SARS / 中华流行病学杂志

<p><b>OBJECTIVE</b>To examine the RNA of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in the blood and excretion of convalescent patient with SARS for prevention and treatment of the disease.</p><p><b>METHODS</b>A total of 276 samples, including plasma, urine, feces and sputum, obtained from 23 convalescent patients with SARS were studied at 3 time-points at least 21 days after the onset of symptoms. RNA was extracted and nested reverse transcription-polymerase chain reaction (RT-PCR) was carried out using SARS-CoV specific primers.</p><p><b>RESULTS</b>Among the 276 samples, SARS-CoV RNA was detected in 6 cases (38.8%) by nested RT-PCR. The positive rates of SARS-CoV RNA was 5.8% in feces and 2.9% in sputum samples but SARS-CoV RNA was not detectable in plasma and urine of all the cases.</p><p><b>CONCLUSION</b>The existence of SARS-CoV RNA in the excretion of some convalescent patients with SARS showed that the excretion from these patients should be carefully treated whilthe re-transmission of SARS by which, should be further studied.</p>

Receptor-binding ability of fragments 260-600 and 397-796 of SARS-associated coronavirus spike protein / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).</p><p><b>METHODS</b>Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.</p><p><b>RESULTS</b>The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.</p><p><b>CONCLUSION</b>Both S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.</p>

Preparation and characterization of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.</p><p><b>METHODS</b>BALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.</p><p><b>RESULTS</b>After cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.</p><p><b>CONCLUSION</b>The anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.</p>