The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

Objective To establish the finite element model of upper cervical vertebrae C0-3 with Jefferson fracture, and to analyze the influence of posterior atlantoaxial fusion (PSF) and occipitocervical fusion (OCF) on biomechanical properties of the vertebral body and mechanical conduction of the screw-rod system. Methods Based on CT images, the C0-3 segment Jefferson fracture model of human upper cervical spine was established. PSF, OCF1 and OCF2 internal fixation were performed according to surgical plan in clinic, and 50 N concentrated force and 1.5 N·m torque were applied to bottom of the occipital bone. The stress distribution and range of motion (ROM) of the cervical vertebral body, the maximum stress of the screw-rod system and the stress distribution of the intervertebral disc for C0-3 segment during flexion, extension, bending and rotation of the upper cervical spine were studied. Results Compared with PSF, the ROM of OCF1 and OCF2 vertebral bodies increased, and the stress of the nail rod decreased. OCF had a better fixation effect. Conclusions PSF, OCF1 and OCF2 fixation method can reduce the upper cervical ROM and restore stability of the upper cervical spine, which make stress distributions of the vertebral body and intervertebral disc tend to be at normal level. The research result can provide a theoretical basis for clinical surgery plan.

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.
Animals ; Chromatography, Affinity ; instrumentation ; Diagnostic Tests, Routine ; standards ; Gold Colloid ; chemistry ; Haemophilus Infections ; diagnosis ; Haemophilus influenzae ; Humans ; Limit of Detection ; Rabbits ; Sensitivity and Specificity

Objective: To investigate the comprehensive nutritional status and diet behavior of middle aged and elderly women with osteoporosis, and thereby to explore the relationship between diet behavior and comprehensive nutritional status. Methods: 311 middle-aged and elderly women with osteoporosis in Chengdu were included in this study. Mini Nutritional Assessment (MNA) was applied to assess their comprehensive nutritional status. Information of social-demographic characteristics and diet behavior (about meals, snacks and water drinking, etc.) of the subjects was collected by questionnaire. Chi square test was used to assess the differences in nutritional status among patients who have different eating behaviors. Logistic regression analysis was performed to evaluate the relationship between diet behaviors and comprehensive nutritional status. Results: The mean MNA score of subjects was 25.8±2.5. 20.3% (63/311) of the subjets were at risk of potential malnutrition, but there was no malnourished subjects found. 46.9% (46/311) of the subjects were in good appetite. 95.2% (296/311) of them had a fixed food intake each meal. 65.8% (198/311) of them had snacks every day, and the most common choice was fruit (86.4% (248/287)). 54.8% (165/311) of them had initiative drinking water habits, and the most common choice was plain boiled water (79.9%, 246/308). 76.5% (238/311) of them had daily portable water less than 1 500 ml. After adjusting the effects of age, occupation and education level, bad appetite (OR=3.50, 95%CI: 1.18-10.62), unfixed food intake (OR=7.27, 95%CI: 1.40-35.83), and seldom or never intake of snack (OR=3.71, 95%CI: 1.42-9.72) were risk factors for malnutrition risk, while tea drinking was protective factor(OR=0.31, 95%CI: 0.11-0.93). Conclusion Risk of potential malnutrition and unhealthy diet behavior among the middle aged and elderly women with osteoporosis should be paid more attention. Unhealtghy diet behavior has a negative effect on their comprehensive nutritional status.

BACKGROUND:Intramedul ary nails and locking plates are widely used for two-part proximal humerus fracture. Which is better for two types of implants in patients remains controversial. OBJECTIVE:To determine the clinical outcome of intramedul ary nails versus locking plates for two-part proximal humerus fracture according to Cochrane Meta analysis. METHODS:We searched PubMed, SCI, Embase, the Cochranel Library and CBMdisc, VIP information, Wanfang Database, and CNKI for randomized control ed trials and quasi-randomized control ed trials on intramedul ary nails and locking plates for two-part proximal humerus fracture. RevMan 5.2 software was used to analyze operation time, intraoperative blood loss, fracture healing time, postoperative complications (heterotopic ossification, pain, screw penetration, necrosis of humeral head), and Constant Score. RESULTS AND CONCLUSION:Six articles of clinical control ed trials were included with 259 patients. 131 patients received intramedul ary nails, and 128 patients received locking plates. Meta-analysis displayed that no significant difference in fracture healing time, heterotopic ossification, pain, necrosis of humeral head and Constant Score was detected between intramedul ary nails and locking plates for two-part proximal humerus fracture. Operation time, intraoperative blood loss, and screw penetration were significantly less in the fixation with intramedul ary nails than that in locking plates (P<0.05). These findings suggested that compared with locking plates, intramedul ary nails method for two-part proximal humerus fractures could reduce screw penetration.

BACKGROUND:J2 takes functional domain (MHC CD4-D1/) of complex conjugate of CD4 molecule and MHC class II molecule as a target, and is a smal molecule compound obtained by computer screening from a chemical data containing hundreds of thousands of organic compounds. In the previous study, J2 was used in mouse models of skin transplantation and keratoplasty by oral and intraperitoneal injection. Results verified that J2 could prolong the survival time of grafts, and suppress occurrence of rejection. To better play the role of a drug targeting and to reduce systemic toxicity, J2 wil be further utilized in local treatment of keratoplasty rejection. OBJECTIVE:To investigate the inhibitory effect of new immunosuppressive agent J2 on CD4+ and CD8+T cel immune functions in rat models receiving alogenic penetrating keratoplasty. METHODS:Alogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and Sprague-Dawley rats as recipients. Group A: normal Sprague-Dawley rats were injected with 0.05 mL placebo subconjunctivaly. Surgery rats were randomly divided into three groups. Group B: alograft rats were injected with 0.05 mL placebo subconjunctivaly after autologous keratoplasty. Group C: alograft rats were injected with 0.05 mL placebo subconjunctivaly. Group D: alograft rats were injected with 1% J2-nanosuspension 0.05 mL subconjunctivaly. The distribution of T cel subsets in peripheral blood was detected using flow cytometry at 3 days, 1, 2 and 3 weeks after transplantation and compared among groups. RESULTS AND CONCLUSION: There was no significant difference in total CD3+ T cels, CD4+ T cels, CD8+ T cels and CD4+/CD8+ in peripheral blood lymphocytes in group B at various time points. At 3 days and 1 week after surgery in group C, no significant difference in total CD3+ T cels, CD4+ T cels and CD8+ T cels was detected. At 1 and 2 weeks, the number of total CD3+ T cels, CD4+ T cels and CD8+ T cels increased, showing significant differences (P < 0.05). In group D, no significant hyperplasy was found in CD4+ T cels and CD8+ T cels at 1 and 2 weeks. The horizontal comparison of the same time point: the total CD3+ T lymphocytes of group D was significantly less than group C at 3 days, 1 and 2 weeks after operation (P < 0.05), whereas there was no significant difference at 3 weeks between the group D and group C. The number of CD4+ T lymphocytes in group D was less than in group C at 3 days and 1 week, but with no significant difference. The ratio of CD4+/CD8+ had no significant difference in group D compared with group C at 3 days, 1 and 3 weeks. J2 inhibits T lymphocyte proliferation and then inhibits T cel-mediated corneal alograft rejection.

BACKGROUND:Some control studies attempt to answer the advantages and disadvantages of high-dose methylprednisolone sodium succinate therapy for acute spinal cord injury in adults, but have arrived at different conclusions. OBJECTIVE:To explore the therapeutic efficacy of high-dose methylprednisolone sodium succinate therapy on acute spinal cord injury in adults by Meta analysis. METHODS:PubMed, Embase, Cochranel Library, CBMdisc, VIP and WanFang Databases were searched by computer, and relevant Chinese and English orthopedic journals were retrieved by hand. Controled trials related to high-dose methylprednisolone sodium succinate therapy of acute spinal cord injury in adults were included. The methodology quality of included trials was criticaly assessed. RevMan 5.0 software was used for data analysis. RESULTS AND CONCLUSION:Nine clinical controled trials were included. Meta-analysis results showed that compared with the conventional therapy, the neurological recovery rate after 24 hours of administration, pneumonia incidence and gastrointestinal reactions increased significantly after high-dose methylprednisolone sodium succinate therapy. However, there were no statistical differences in the rate of urinary tract infection, nonunion rate and stress ulcer incidence between these two therapies. These findings indicate that the high-dose methylprednisolone sodium succinate therapy on acute spinal cord injury in adults has better outcomes in neurological function recovery, but can lead to higher incidence of lung infection and gastrointestinal reactions. Therefore, lung infection and gastrointestinal reactions should be avoided as much as possible during the course of treatment.

Objective_To study the effect of human insulin on cell cycle progression and apoptosis of rat liver cell line BRL-3A in vitro.Methods_MTT method was used to observe the effect of insulin on cell activity, and flow cytometry was used to detect cell apoptosis and cell cycle.qRT-PCR was used to evaluate the expression of related genes.Results_Human insulin induced the proliferation of BRL-3A cells in a dose-dependent manner ( P<0.05 or P<0.01);After 3 days treated by human insulin (500 nmol/L), the proportion of cells in G0/G1 phases re-markably decreased (P<0.05).Moreover, pro-apoptotic BAX was down-regulated (P<0.05), while cell prolif-eration-related gene CCNA2 was up-regulated (P<0.05).Conclusions_Human insulin may inhibit the apoptosis of BRL-3 A cell line and induce proliferation due to the down-regulated expression of BAX and up-regulated expres-sion of CCNA2 .

BACKGROUND:Intramedul ary nail and traditional steel plate fixation have been widely used in the treatment of distal tibial (extra-articular) fractures, but these two kinds of methods have shortcomings. Some control studies attempt to answer the advantages and disadvantages of the issue on the treatment of extra-articular distal tibial fractures with intramedul ary nail and steel plate fixation, but the conclusions are different.
OBJECTIVE:To determine the efficiency and safety of intramedul ary nail and plate fixation in the treatment of extra-articular distal tibial fractures by Meta analysis.
METHODS:PubMed, Embase, Cochrane Library, CBMdisc, Weipu information database and Wanfang were searched by computer, and relevant Chinese and English orthopedic journals were hand-searched. Control ed trials related to intramedul ary nail and plate fixation in the treatment of extra-articular distal tibial fracture were included. The quality of trials was critical y assessed. RevMan 5.0 software was used for data analysis.
RESULTS AND CONCLUSION:Four articles were included. Meta analysis showed that compared with the plate fixation, the malunion rate of the intramedul ary nail for extra-articular distal tibial fractures increased. There was no statistical difference in the rate of nonunion rate, delayed fracture healing, and infection rate after operation. These indicate that intramedul ary nail and plate fixation both have good effects on fracture healing.