The Prokaryotic Expression And Polyclonal Antibody Preparation Of The Main B Cell Antigen Epitopes Of DEV UL53 Gene And The Primary Application Of The Prepared Polyclonal Antibody

The essay focuses researches on the sequence characteristics, prokaryotic expression and polyclonal antibody preparation of the main B cell antigen epitope of DEV UL53 gene (GenBank Accession No.:EU071035), the subcellular localization of gK and so on. The main researching results such as follows:1. The bioinformatics analysis of UL53 gene The ORF of DEV UL53 gene is 1032bp and encoding 343 amino acid residues, which is 38.119KDa in theoretical molecular weight. Through the prediction of GENESCAN on line, the result indicates the integral ORF of DEV-UL53 gene could be divided into two parts, which contained 1 to 675 bp optimal exon domain and 676 to 1032 bp suboptimal exon domain. The predicted results by informatics indicate that the putative protein has an N-terminus signal sequence cleavage site at amine acid residue 30, three potential N-linked glycosylation sites, eleven phosphorylation sites and four transmembrane domains. The results of amino acid sequence similarity analysis and phylogenetic evolution tree illustrate DEV UL53 gene has a closer evolutionary relationship with fowl Alphaherpesvirus containing gallid herpesvirus 2 and gallid herpesvirus 3. Moreover, the predicted result of subcellular localization demonstrates that the gK mainly locates at cytoplasmic membrane. The codon usage bias analysis of DEV UL53 gene describes the bias codons have strong bias towards A-ended or T-ended codon at the third codon position.2. The expression plasmid construction, prokaryotic expression of UL53 gene and preparation of polyclonal antibody According to the predicted sequence characteristics of UL53 gene, it's inferred that DEV UL53 gene is not easy to express. Thus, we combine some parameters (B cell epitopes, optimal exon domain, transmembrane domains) together to design another pair of primers for DEV truncated UL53 gene, which is also the main B cell epitopes of UL53 gene. The two pairs of primers are used to amplify the UL53 gene and truncated UL53 gene(tUL53) respectively. And then we begin to T clone and construct the expression plasmids (pET-32b(+)/UL53,pET-32b(+)/tUL53). Through IPTG induction expression, the result indicates that the DEV UL53 gene does not express, while the truncated UL53 gene can express well in prokaryotic expression system. The result of experiment is consistent with that of prediction. The optimized expression condition is 0.4 mmol/L IPTG and inducing 8h at 37℃, meanwhile the expression product exists as inclusion body. The expression product is identified as one of proteins encded by DEV with rabbit anti_DEV whole serum through Western blot. And next, the purified protein is used to prepare polyclonal antibody. The titer of prepared polyclonal antibody is 1:32, which is detected by agar gel diffusion test; the neutralization titer of the rabbit anti_tgK polyclonal antibody is 1:5.623.3. Identification early or late gene of UL53 gene①UL53 gene transcription phase:the UL53 gene transcript is firstly detected at 10h post infectionem, which detects the transcript at different time by real-time fluorescent quantitation PCR. The amount of UL53 gene transcript is increasing with the time, reaches the highest point at 36h post infectionem and decreases at 48h post infectionem;②The nucleic acid inhibition test further identifies DEV UL53 gene is late gene;③UL53 protein expression phase:the protein encoded by UL53 gene is primarily detected at 14h post infectionem in infected DEF through Western blot. Compared with the prevenient outcomes, it's demonstrated the DEV UL53 gene, which encodes the Glycoprotein K, belongs to the late gene.4. The subcellular localization and tissues localization of UL53 gene product The artificial infected DEV cells and tissues were detected by indirect immunofluorescence. The results indicate:①The DEV UL53 gene product is detected at 12h post infectionem in the cytoplasm of infected cells;②the localization analysis of DEV UL53 gene product in artificial infected DEV tissues illustrates the manifest positive signals can be found in Harder's glands, thymus, liver, kidney, lung, cecum, duodenum and rectum; while in spleen and cardiac muscle, the weak positive signals can also be detected.