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Objective To express and preliminary identify scavenger receptor B2 (SCARB2)protein.Methods SCARB2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR)from mRNA extracted from RD cells,and cloned into pMD19-T vector.Expression vector pET28a(+)/SCARB2 was constructed,and SCARB2 protein was expressed in prokaryotic ex-pression system.Obtained recombinant proteins were identified by detection of Western blot using His labeled monoclonal antibody.Results Recombinant SCARB2 proteins,expressed by induction of isopropy-β-D-thiogalactoside (IPTG),were success-fully purified and specifically recognized by His labeled monoclonal antibody.Conclusion SCARB2 proteins could be expressed and identified successfully,which could provide reference for researching mechanism of enterovirus type 71 (EV71)and scavenger re-ceptor protein,and for preparation of SCARB2 monoclonal antibodies.
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Objective To study the genomic molecular organization and genogroup of human metapneumovirus(hMPV) infected infants in Guangzhou of China. Methods Primers were designed on the basis of the genomic sequence of hMPV 00-1 strain(AF371337) in the GenBank, and amplify hMPV genomeby RT-PCR. The PCR-products were cloned to T vector and sequenced, the genomic nucleotide sequences were analyzed with the programs Clustal W/X, DNASTAR and MEGA4. 1. Results The cloned strainhMPVgz01 genome is 13 327 bp in length, the genome contains eight open reading frames in the order 3-N-P-M-F-M2-SH-G-L-5. The genomic sequences of hMPVgz01 strain are compared with those of hMPV in GenBank, revealed that the homology with hMPV group A ranges between 92%-97%, homology with group B is 81%, and with avian metapneumovirus group C is 71%, the highest homology is with BJ1887 strain of genogroup A2b. The N, F, G genes of hMPVgz01 strain are compared with those corresponding genes of hMPV subgroups A1, A2, B1, B2, revealed that the highest homology is also with genogroup A2b. Conclusion The complete nucleotide sequence of hMPVgz01 strain isolated from Guangzhou in China is 13 327 bp in length, GenBank accession No. is GQ153651. Comparison of the genomic sequence and three genes of hMPVgz01 strain with those corresponding sequences of hMPV show the highest homology is with genogroup A2b. Sequence and phylogenetic analysis of the hMPVgz01 strain revegled that this isolate belongs to genogroupA2b.
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Objective To develop and verify an influenza A virus antigen-detecting kit which can detect all the subtypes of influenza A virus. Methods Double-antibodies sandwich enzyme linked immunosorbent assay (ELISA) was utilized for developing the influenza A virus antigen-detecting kit. The sensitivity, specificity, accuracy and stability of the kit were evaluated by the clinical samples. Results The lower detection limit of the kit for the N protein was 7. 63 ng/mL, which was 256 times lower than that of the hemagglutination and 16 times lower than that of immune colloidal gold technique. The kit didn't show any cross-reaction with the influenza B virus, respiratory syncytial virus, respiratory adenovirus, para-influenza virus type Ⅰ and Ⅲ, mycoplasma pneumomiae, avian newcastle disease virus, avian infectious bursal disease virus or avian infectious bronchitis virus. The specificity was 100%. Both the intra batch variaton coefficient (CV) value and inter batch CV value were less than 15%, which met the national standard for ELISA kits. The results proved that the kit could keep stable at 4 ℃ for more than 1 year and at 37 ℃ for more than 7 days. The kit could identify H1N1, H3N2, H5N1 and H9N2 influenza A viruses. The clinical research data of human influenza virus showed the consistency rate between the kit and regular cell culture method was 93. 44% for the positive samples and 99. 31% for the negative samples. The clinical research data of avian influenzavirus showed the consistency rate between the kit and regular cell culture method was 95. 45% for positive samples and 98. 09% for negative samples. Conclusion The influenza A virus antigen-detecting ELISA kit can be used for the epidemiological survey of the infection of human influenza A virus or avian influenza virus with high sensitivity and specificity.