Objective:To prepare recombinant protein of group 36 allergen of Dermatophagoides farinae(Der f 36),to deter-mine its immunogenicity and bioinformatics analysis was performed.Methods:Nucleic acid sequence coding for Der f 36 was obtained and artificially synthesized,pET-28a(+)was inserted to construct pET-28a(+)-Der f 36 plasmid and transformed into BL21(DE3)receptor cells.After expressed and purified in BL21(DE3),recombinant allergen rDer f 36 was obtained,recombinant protein rDer f 36 was identified by SDS-PAGE and Western blot.Serum IgE binding rates of rDer f 36 was determined by IgE-ELISA.Cross-reactivity between rDer f 36 and rDer p 36 was detected by IgE-ELISA inhibition assay.HNEpC cells were cultured with rDer f 36 for 24 h and cytokines were detected.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 36 and Der p 36.Results:Coding gene for Der f 36 was obtained with a total length of 690 bp,whose molecular weight was 25.6 kD;serum IgE binding rate of rDer f 36 was 42.1%by IgE-ELISA;IgE-ELISA inhibition assay showed that rDer p 36 had 40%(8/20)inhibition rate(>50%)for rDer f 36,with an average inhibition rate of 52.98%.Compared with control group,HNEpC cells cultured with rDer f 36 showed that IL-6,IL-8,IL-33,IL-25 and TSLP expressions had increased;bioinformatics analyses show that sequence consistency of Der f 36 and Der p 36 was 77.63%,with similar physicochemical properties.Secondary structure analysis showed that both of them contained α helix,β-turn and random coils,and content of random coils was the highest;structure of C2 domains was also highly overlapping(RMSD=0.046).Conclusion:Recombinant allergen rDer f 36 is successfully prepared with good immunogenicity,laying foundation for diagnosis and treatment of dust mite allergen single component.High similarity in physical and chemical properties,secondary structure,and tertiary structure between Der f 36 and Der p 36 determines their cross reactivity.

Objective:To prepare recombinant protein of group 27 allergen of Dermatophagoides farinae(Der f 27),and to determine its immunoactivity.Methods:pET-28a(+)-Der f 27 plasmid was constructed and inserted into E.coli BL21(DE3)cells.After being expressed and purified,recombinant allergen rDer f 27 was obtained.IgE binding rates of rDer f 27 with sera from patients with allergic rhinitis induced by Dermatophagoides farinae was determined by ELISA and Western blot.PBMC from patients with allergic rhinitis and BESA-2B cells were cultured with rDer f 27 for 24 h,respectively,and cytokine expression was measured.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 27.Results:pET-28a(+)-Der f 27 plasmids were prepared successfully and transformed into BL21(DE3)cells.After expressed and purified with PTG,SDS-PAGE and Western blot identified a band about 48 kD.IgE binding rates of rDer f 27 were 39.5%and 45.5%with sera from patients with allergic rhinitis allergic to Dermatophagoides farinae by IgE-ELISA and IgE-Western blot,respectively.Compared with control group,IL-6 and IL-8 expressions were increased in PBMC from patients with allergic rhinitis being cultured with rDer f 27(P<0.05);expressions of IL-10 and TGF-β were decreased in BESA-2B cells being cultured with rDer f 27,while IL-17A and IL-23A expressions were increased.Bioinformatics analysis showed that Der f 27 belong to serine protease inhibitor family and had universal structure and func-tion of this family.Secondary structure of Der f 27 was mainly composed of α-helix(42.62%)and random coil(35.60%).Conclusion:Recombinant allergen rDer f 27 has been prepared successfully with good immunoreactivity and immunogenicity,becoming one of important allergens of allergic rhinitis.

Objective:To establish a quantitative detection method for the main components of dust mite allergens Der p 1, Der p 2 specific immunoglobulin E (sIgE) by using the nano-magnetic particle chemiluminescence immunoassay.Methods:The performance indexes of the established method were evaluated after setting up and optimizing the chemiluminescence detection system and immune reaction conditions of sIgE for dust mite allergen. Serum sIgE levels of 50 suspected allergic patients with dust mite were determined by this chemiluminescence method. At the same time, this method was compared with the Phadia kit and the consistency was analyzed by Kappa test. Results:The optimal amount of magnetic beads was 25 μg, the optimal reaction buffer (pH=7.4) contained 0.1 mol/L Tris-HCl and 0.25%( W/ W) casein, the optimal coating solution contatined 20 mmol/L phosphate buffer (PB) and 1%( W/ W) bovine serum albumin (BSA), and the luminescence enhancement solution contained 0.05%( V/ V) Triton X-100. The two-step immunoreaction was adopted, and the detection could be completed with 20 μl sample at the optimal reaction temperature of 37℃. The limit of detection (LOD) of the established nano-magnetic particle chemiluminescence system in detecting Der p 1 and Der p 2 sIgE antibodies were both less than 0.01 kU/L, with the linear range of 0.2-100.0 kU/L, the precision of less than 7%, and the cross contamination rate of 0.19% and 0.21%. Compared with the Phadia system, the positive and negative coincidence rate of Der p 1 were 78.0%(32/41) and 9/9 with good consistency ( Kappa=0.65, P=0.008), and the positive and negative coincidence rate of Der P 2 were 93.3%(28/30) and 85.0%(17/20) with good consistency ( Kappa=0.79, P=0.003). Conclusion:The nano-magnetic particle chemiluminescence immunoassay is successfully established for detecting dust mite allergen sIgE, which has good detection performance and good consistency with Phadia system.

Bronchial asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and recurrent episodes of reversible airway obstruction. Due to different pathophysiological mechanisms, the disease is very heterogeneous in clinical manifestation, course of disease, response to treatment, phenotype etc. There is a strong need for biomarkers to assess the characteration and severity of the disease. Recently, lymphocyte and blood cells, antibodies, cytokines, chemokines, noncoding RNA and other protein markers have been studied as blood biomarkers of asthma. The present article summarized these biomarkers in diagnosis, phenotyping and treatment efficacy.

In the past ten years, the research and application of microbiome has continued to increase. The microbiome has gradually become the research focus in the fields of life science, environmental science, and medicine. Meanwhile, many countries and organizations around the world are launching their own microbiome projects and conducting a multi-faceted layout, striving to gain a strategic position in this promising field. In addition, whether it is scientific research or industrial applications, there has been a climax of research and a wave of investment and financing, accordingly, products and services related to the microbiome are constantly emerging. However, due to the rapid development of microbiome sequencing and analysis related technologies and methods, the research and application from various countries have not yet unified on the standards of technology, programs, and data. Domestic industry participants also have insufficient understanding of the microbiome. New methods, technologies, and theories have not yet been fully accepted and used. In addition, some of the existing standards and guidelines are too general with poor practicality. This not only causes obstacles in the integration of scientific research data and waste of resources, but also gives related companies unfair competition opportunity. More importantly, China still lacks national standards related to the microbiome, and the national microbiome project is still in the process of preparation. In this context, the experts and practitioners of the microbiome worked together and developed the consensus of experts. It can not only guide domestic scientific research and industrial institutions to regulate the production, learning and research of the microbiome, the application can also provide reference technical basis for the relevant national functional departments, protect the scale and standardized corporate company's interests, strengthen industry self-discipline, avoid unregulated enterprises from disrupting the market, and ultimately promote the benign development of microbiome-related industries.
China ; Consensus ; Humans ; Industry ; Microbiota

Objective: To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen. Methods: According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method. Results: In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml.In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen. Conclusion A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.

Objective:To obtain the prokaryotic expression product for the group 6 allergen of Dermatophagoides farine ( Der f 6) and detect its IgE-binding rates with sera from asthmatic children. Methods: By enzyme digestion of pET28a (+)-Der f 6 with BamHⅠ plus XhoⅠ,the target gene Der f 6 was obtained and linked into the vector pET32a (+) to construct the recombinant plasmid pET32a(+)-Der f 6, which was then transfected into E. coli BL21 cells for expression, induced with isopropyl-β-D-thiogalactoside ( IPTG) ,purified by affinity chromatography and identified by SDS-PAGE,Western blot and AMLDI-TOF,and tested by ELISA for IgE reactivity with sera from asthmatic children. Results:The plasmids pET32a(+)-Der f 6 were constructed,transformed into E. coli BL21 and expressed successfully. SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and the His-tag in the plasmids,and MALDI-TOF/TOF identified the identical structure to the allergen Der f 6. Using the ELISA method developed with the recombinant proteins as coating antigen,the positive rate was 41. 3% (19/46) in asthmatic children allergic to dust mite. Conclusion: The plasmids pET32a (+)-Der f 6 were constructed successfully,expressed in E. coli BL 21 (DE3). The recombinant fusion protein has a good reactivity with sera from asthmatic children.

Objective:To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody.Methods: The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a(+)-Der f 7.BALB /c mice were immunized with the recombinant protein.Myeloma cells and spleen cells were fused,and hybridoma cells were screened by ELISA.Hybridoma cells were injected into the mice abdominal cavity to obtain ascites.It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody.The specificity of the antibody was identified by Western blot.Results: Three hybridoma cells which stably secret recombinant Der f 7 monoclonal antibody were obtained.The monoclonal antibody had high purity,the titer was higher than 1∶243 000.Western blot showed that Der f 7 recombinant protein could be recognized well.Conclusion: We successfully obtained Der f 7 monoclonal antibody,which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.

OBJECTIVE: To explore the eff ect of artenisiae scopariae and poriae powder (ASPD) on calpain-2 expression in liver tissue from rats with obstructive jaundice. METHODS: The rat model of obstructive jaundice was established. SD rats was divided into the control group, the obstructive jaundice group, the obstructive jaundice model plus ASPD group, the obstructive jaundice model plus saline group. Th e serum levels of TBIL, ALT, AST and other biochemical indexes were detected. The pathological changes of liver tissue were evaluated by HE staining. The calpain-2 mRNA and protein expression in liver was measured by Real-time PCR and immunohistochemistry or Western blot, respectively. RESULTS: The calpain-2 mRNA and protein expression levels were significantly up-regulated in live tissues from the rats with obstructive jaundice in a time-dependent manner. The ASPD could inhibit the calpain-2 expression in rats with obstructive jaundice concomitant with the decreased liver damage and the improved liver function, suggesting that calpain-2 was involved in endoplasmic reticulum stress-mediated cellular apoptosis and the occurrence of obstructive jaundice. CONCLUSION ASPD could be used for patients with obstructive jaundice to promote the recovery of liver function after operation and to reduce the incidence of complications, which provide a theoretical basis for the reasonable application of traditional Chinese medicine in the peroperative period.
Animals ; Apoptosis ; Artemisia ; chemistry ; Calpain ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; Jaundice, Obstructive ; enzymology ; Liver ; drug effects ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.