A preparation method and application of gene engineering expression qx type chicken infectious bronchitis virus s1 protein antigen
A protein and antigen technology, applied in the application field of detecting QX type IBV antibodies, can solve the problems that the antibody level does not have serotype specificity, cannot apply serotype specific immune antibodies, evaluation, etc.
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Embodiment 1Q
[0029] The bioinformatics analysis of embodiment 1QX type IBV S1 gene
[0030] Taking the QX-type IBV vaccine strain QXL87 as the analysis object, first, according to the S1 gene sequence of the strain (GenBank accession number: MH743141; SEQ ID NO.1), the MegAlign program in the BioEdit7.0 and Lasergene 7.0 software packages was used, etc. With Mass type H120 strain (GenBank accession number: FJ888351.1; SEQ ID NO.2), 793 / B type 4 / 91 strain (GenBank accession number: AF093793.1; SEQ ID NO.3) and tl / ch / LDT3 / 03 type LDT3-A strain (GenBank accession number: KR608272.1; SEQ ID NO.4) and other sequences were compared for nucleotide and amino acid sequences, and then the protein sequence was analyzed by the Protean program in the Lasergene7.0 software package. After comprehensive comparison, 5 polypeptide fragments A-E located in the hypervariable region of S1 and with good antigenicity were selected for further screening. The amino acid sequences corresponding to the 5 polypeptid...
Embodiment 2
[0033] Embodiment 2: Expression of 5 polypeptide fragments of A~E in Escherichia coli
[0034] Primer design and PCR amplification of target fragments: According to the 5 polypeptide fragments screened out from A to E, determine the corresponding IBVQXL87 strain S1 gene coding region sequence, followed by S1-A (SEQ ID NO.10), S1-B (SEQ ID NO.11), S1-C (SEQ ID NO.12), S1-D (SEQ ID NO.13), S1-E (SEQ ID NO.14). 5 pairs of specific primers were designed according to the gene sequence of each segment. For the convenience of cloning, the BamH I restriction site recognition sequence was added to the 5' end of the upstream primer, and the Xho I restriction site recognition sequence was added to the 5' end of the downstream primer. For the primer sequence, see Table 2. Using the genomic RNA of the QXL87 strain as a template, 5 target fragments were amplified by RT-PCR method, and the electrophoresis identification results of the PCR products are shown in figure 2 . The five target ...
Embodiment 3
[0042] Embodiment 3: Carry out ELISA detection to QX type IBV positive serum with purified recombinant protein as coating antigen
[0043] 5 kinds of recombinant proteins S1-A, S1-B, S1-C, S1-D and S1-E were coated with 100 μL / well of 2 μg / mL on a 96-well microtiter plate, and the QX type IBV (QXL87 strain) was immunized The multiple antiserum prepared from SPF chickens was diluted 1:100, and the ELISA test was carried out with 5 kinds of antigen-coated microtiter plates, and the antigen with the best detection effect on QX-type specific antibodies (the largest P / N value) was selected for use. To establish a method for the detection of QX-type IBV-specific antibodies. The specific operation steps are as follows:
[0044] (1) Preparation of QX type IBV positive serum
[0045] SPF chickens were immunized for the first time at the age of 14 days, and the QX type IBV vaccine strain QXL87 strain (10 6.5 EID50 / ml) is preserved and provided by the Key Open Laboratory of Livestock ...
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