Cloning,Expression And Immunological Identification Of LOOP1-6 Protein Of Human Adenovirus Type 3

ObjectivesThe typical representative strains of human influenza virus B were screened by polymerase chain reaction experimental technique and their homology with GenBank was discussed. According to the detection results, a representative strain was selected to construct a plasmid encoding LOOP1-6 protein and the purified LOOP 1-6 target protein was purified in E.coli BL21 and immuno logical identification by Weston-Blot. Through inquiry of variation and changes for the capsid protein gene and LOOP 1-6 target protein was obtained through the recombinant plasmid was successfully cloned, expressed, purified and immunological identification, they provide experimental guidance for appropriate clinical serological diagnosis kit and adenovirus infection prevention and control.Methods1. Using the production of ABT9+7 respiratory viruses by multiplex PCR rapid detection reagent of Beijing Zhuo Cheng hui sheng Biological Technology Company to detect pharyngeal swabs of suspected cases of children clinical upper respiratory tract viral infection for finishing the work of preliminary screening of the virus or by Ou Meng medical laboratory diagnostics stock company respiratory tract pathogens spectrum antibody IgM detection reagent box (indirect immunofluorescence assay) to detect level of the serum antibody.2. According to the LOOP1-6 or LOOP7 gene of adenovirus hexon can be set in the serum type, design the corresponding primers, PCR and sequencing. By sequencing the results, a sequence of Blast was performed on the NCBI, to determine the type of adenovirus.3. Choicing 3 types of adenovirus, isolation and proliferation of adenovirus. After the proliferation was completed, the nucleic acid was extracted. According to the GenBank, Penton base, the corresponding primers of fiber and hexon gene sequences of adenovirus type 3 were designed for PCR amplification and sequencing.4. Sequencher 5.0 was used to deal with the results of sequencing. The phylogenetic relationships of the processed sequences were analyzed by software MEGA5.0, and the amino acid and nucleotide homology analysis was carried out.5. Choicing and cloning Hexon gene LOOP 1-6 of adenovirus type 3 to carrier plasmid pET-48b (+), It was transformed into E. coli BL21 (DE3), induced by IPTG and by nickel ion affinity chromatography purification, to get target protein LOOP 1-6. The expression of protein was identified by Weston-Blot method with anti 6xHis-tag antibody.Results1. From Shaanxi, Hunan, Hebei, Shandong, Yunnan, Gansu and Jilin seven provinces, the results of the specimens adenovirus detection were 229 pieces which was consisted of 121 copies of adenovirus type 7,35 copies of adenovirus type 3 and 73 copies of other types.2. In the phylogenetic tree, during the 12th Five Year Plan period, the HAdV 3 gene of the seven provinces was generally consistent with Worldwide popular profiles. Two recombinant strains of Hexon gene were found in Hunan, which were respectively collected in 2012 and 2013. The two strains and Japan separation P7H3+7F3 strain (genbank:JN860679.1) in 2011 nucleotide homology was from 98.3% to 98.4% and amino acid identity was from 99.6% to 99.6% in hexon gene, nucleotide homology was from 99.2% to 99.2% and the homology of amino acid was 98.7% in Penton base gene, nucleotide homology was 63.6% and the amino acid homology was 57.1% in fiber gene(as shown in figure 8-A. B and C). In this laboratory, they are named P7H3+7F7 strain, which shows that the hexon gene of HAdV 3 has the possibility of recombination within the group. Apart from these two strains, other strains nucleotide homology was from 99.7% to 100% and amino acid homology was 100% in fiber gene, nucleotide homology was from 99.9% to 100% and amino acid homology between 99.7% and 100% in hexon gene, nucleotide homology was from 98.8% to 100% and amino acids homology was from 98.3% to 100% in Penton base gene. I n summary, The HAdV 3 gene is very conservative.3. The recombinant plasmid was successfully cloned and expressed, and the purified LOOP 1-6 target protein was obtained.ConclusionUsing multiplex PCR and indirect immunofluorescence of serum successfully identified the adenovirus infection positive sepcimens in this study, sepcimens reamed poison after the success, the sequencing of PCR was discussed in this paper in seven provinces of China during the 12th Five Year Plan, the capsid protein gene of adenovirus type 3 is more conservative. The recombinant plasmid was successfully cloned and expressed, and the purified LOOP1-6 target protein was obtained, which provided a preliminary experimental basis for the further study of the serum typing kit in the later stage.