Evaluation of immunoprotection against coronavirus disease 2019:Novel variants,vaccine inoculation,and complications / 药物分析学报

The strikingly rapidly mutating nature of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)genome has been a constant challenge during the coronavirus disease 2019(COVID-19)pandemic.In this study,various techniques,including reverse transcription-quantitative polymerase chain reaction,antigen-detection rapid diagnostic tests,and high-throughput sequencing were analyzed under different scenarios and spectra for the etiological diagnosis of COVID-19 at the population scale.This study aimed to summarize the latest research progress and provide up-to-date understanding of the methodology used for the evaluation of the immunoprotection conditions against future variants of SARS-CoV-2.Our novel work reviewed the current methods for the evaluation of the immunoprotection status of a specific population(endogenous antibodies)before and after vaccine inoculation(adminis-tered with biopharmaceutical antibody products).The present knowledge of the immunoprotection status regarding the COVID-19 complications was also discussed.Knowledge on the immunoprotection status of specific populations can help guide the design of pharmaceutical antibody products,inform practice guidelines,and develop national regulations with respect to the timing of and need for extra rounds of vaccine boosters.

Molecular immune pathogenesis and diagnosis of COVID-19 / 药物分析学报

Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of SARS-CoV-2 has been marked as the third introduction of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coro-navirus (MERS-CoV) in the twenty-first century. In this minireview, we provide a brief introduction of the general features of SARS-CoV-2 and discuss current knowledge of molecular immune pathogenesis, diagnosis and treatment of COVID-19 on the base of the present understanding of SARS-CoV and MERS-CoV infections, which may be helpful in offering novel insights and potential therapeutic targets for combating the SARS-CoV-2 infection.

Prokaryotic expression and purification of the cDNA of recombinant human cytokeratin 9 / 西安交通大学学报(医学版)

Objective To clone and fuse the cDNA of human cytokeratin 9 in prokaryotic expression system,and purify and identify the fusion protein.Methods The cDNA fragment of human cytokeratin 9 was amplified from human keratinocyte (HaCaT) total RNA with specific primers.The PCR products were cloned into vector pET-28a,then the fusion protein of his-CK9 was induced by IPTG.The expressed fusion protein of his-CK9 was purified by nickel ion affinity chromatography and identified by SDS-PAGE and Western blot.Results The sequencing proved that the recombinant vector of the cDNA of CK9 was correct.The fusion protein of his-CK9 was induced to be expressed in E.coli.The fusion protein of his-CK9 was highly purified and his-CK9 showed specific binding to the commercialized antibodies of CK9.Conclusion The recombinant vector of pET-28a-CK9 has been successfully constructed,and the fusion protein of his-CK9 has been successfully expressed and purified.

Feasibility evaluation of real-time quantitative PCR with small reaction volume / 西安交通大学学报(医学版)

Objective To evaluate the feasibility of real-time quantitative PCR (RtPCR) with small volume regarding the stability, efficiency and reliability of amplification, and determine the optimal quantity of cDNA template suitable for small PCR volume. Methods The experiment was carried out in 3 groups with 10, 15 and 20μL reaction volume, respectively. In each group, rat β-actin mRNA was detected by RtPCR with 0.1, 0.2, 0.3 or 0.4μL rat cDNA as template, respectively. The amplification curve and melting curve were used to evaluate the reaction stability. The fitting of a curve of gradient templates against threshold cycle numbers was to show the reaction efficiency and the linear correlativity was to estimate the suitability of the template quantity. In addition, in order to estimate reliability, pristane-induced arthritis (PIA) rat model was established, and spleen TNF-α mRNA expression was detected by RtPCR with the selected reaction volumes. Results The amplification of rat β-actin mRNA was specific and stable in 10μL, 15μL and 20μL PCR volume, and had a high efficiency. Furthermore, the standard curves fitted by 0.1-0.4μL gradient templates showed a significant linear correlation in each volume group. When the 10μL and 20μL PCR volumes, and 0.2μL cDNA templates were chosen, the TNF-α mRNA expression in PIA rat spleen showed significant upregulation in both two volume groups as anticipated. Conclusion The experiment shows that it is feasible in the RtPCR amplification to use the small reaction volume of 10μL and 15μL, which has good stability and reliability. And 0.1-0.4μL templates are all suitable for the reaction system. PCR with small volume can not only save the reagents and template, especially rare clinical specimens, but also is helpful for the realization of high-throughput reaction.

Disturbance of nivalenol on expression of adhesion molecule CD44 on surface of cultured chondrocytes / 中国组织工程研究

BACKGROUND: Deficit of nivalenol (NIV) and selenium (Se) is related with kashin-beck disease (KBD) to certain extent. Hyaluronic acid (HA) metabolism affects directly the polymerization of proteoglycans (PG) and normal structure and function of cartilage. The integration with HA receptor on surface of cartilage tissue is the key link in HA metabolism. Being the main receptor of HA on chondrocytic membrane, CD44 expression impacts directly HA metabolism, further affects cartilage matrix metabolism, which is extremely important to maintaining the structure and function of cartilage matrix.OBJECTIVE: To probe into the injury and protection of related etiology of KBD to target tissue cells and the mechanism on degenerative necrosis of chondrocytes.DESIGN: Randomized controlled observation was designed.SETTING: Department of Genetics and Molecular-Biology of Medical College of Xi' an Jiaotong University.MATERIALS: The experiment was performed in Key Laboratory Room on Ministry of Education associated with Environment and Disease of Xian Jiaotong University from October 2002 to July 2004. One New Zealand pedigree young rabbit aged 30 days was employed and its humerus, femurs and tibia were cut out in surgery.METHODS: With cell culture, the model of bone tissue was reconstructed in vitro, in which, NIV of various concentrations, KBD suspicious infectious agent and Se, the protective factor were added. HA receptor CD44 on chondrocytic membrane and soluble CD44 in cell culture solution were determined.MAIN OUTCOME MEASURES: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface. ② Soluble CD44 in chongrocytic culture solution.RESULTS: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface: CD expression in chondrocytic membrane was decreased with increasing of NIV concentration and it was in tendency of increasing with Se added. ② Soluble CD44 in chongrocytic culture solution:The concentration of soluble CD44 in cell culture solution was decreased gradually following the increased concentration of NIV, but it was increased in high concentration group and such tendency did not alter when Se added. Except blank control and Se control, significant difference was presented among groups (P ＜ 0.05).CONCLUSION: NIV disturbs adhesion molecule CD44 expression on chondrocytic surface and further induces metabolic disturbance of cartilage extracellular matrix. Se supplementation can resist the injury of NIV to chondrocytes, but its action is limited.

Expression and significance of toll-like receptor 4 by peripheral blood mononuclear cell in rats with adjuvant-induced arthritis / 西安交通大学学报(医学版)

Objective To demonstrate the role of innate immunity reaction in rheumatoid arthritis and other autoimmune diseases by studying the expression of toll-like receptor 4(TLR4) by peripheral blood mononuclear cell(PBMC) in rats with adjuvant-induced arthritis(AIA) and analyzing the relationship between expression of TLR4 by PBMC and score of arthritis severity.Methods Both the proportion of CD14+TLR4+ cells in CD14+ cells and the mean intensity of fluorescence(MIF) of TLR4+ cells were analyzed by flow cytometry.Expression of TLR4 mRNA by PBMC was determined by Northern blot.CD14+TLR4+/CD14+ ratio,TLR4 MIF and expression of TLR4 mRNA by PBMC in the model group at 15 days after AIA were compared,respectively,with normal control group and model group at 25 days.The correlation between TLR4 MIF and score of arthritis severity was analyzed by Mann-Whitey U test.Results CD14+TLR4+/CD14+ ratio,TLR4 MIF and TLR4 mRNA expression in model group at 15 days after AIA were obviously increased compared with those in control and model groups at 25 days(P0.05).It indicated that TLR4 participated in the pathogenesis of rheumatoid arthritis.Conclusion TLR4 expressed by PBMC is closely associated with pathogenesis and pathophysiology of rheumatoid arthritis,which suggests that innate immunity response plays an important role in pathogenesis of RA and other autoimmune diseases.