Indirect ELISA detection method for constructing Guertu virus antibody and application
A detection method and antibody technology, applied in the fields of molecular biology and microbiology, can solve the problem that the establishment of GTVNP protein has not yet been reported, and achieve the effects of low-cost technical support, high sensitivity, and accurate and reliable results.
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Embodiment 1
[0051] Example 1: Indirect ELISA detection method for Guertu virus antibodies
[0052] The present invention uses the isolated Guertu virus DXM strain NP sequence for bioinformatics analysis, and connects the designed NP gene with KpnI and NotI restriction sites into the pET-32a(+) empty vector, and the pET -32a-NP, transformed into BL21 competent cells and cultivated in large quantities to obtain rNP protein.
[0053] The present invention specifically provides an indirect ELISA detection method for Guertu virus antibodies, the specific steps are as follows:
[0054] (1) Connect the synthetic NP gene with KpnI and NotI restriction sites into the empty pET-32a(+) vector, and transform the pET-32a-NP vector into E.coli BL21 competent cells.
[0055] (2) Add the E.coli BL21 competent cells prepared in step (1) to the ampicillin (A + After mass propagation overnight in the medium of ), IPTG inducer was added at a ratio of 1:1000, and then cultured for 4 hours.
[0056] (3) Centrifuge the ...
Embodiment 2
[0062] Example 2: Preparation of prokaryotic expression vector pET-32a-NP
[0063] The NP sequence of GTV DXM strain was selected for bioinformatics analysis, and the designed NP gene with KpnⅠ and NotⅠ restriction sites was sent to Shanghai Jierui Bioengineering Co., Ltd. for synthesis, and the synthesized NP gene was ligated into KpnⅠ and NotⅠ The digested pET-32a(+) empty vector was transformed into BL21 competent cells, and the pET-32a-NP was sequenced by Shanghai Bioengineering Co., Ltd. The analysis shows that there is no signal peptide and transmembrane region in the NP sequence of GTV DXM strain. The recombinant plasmid pET-32a-NP was digested with KpnI and NotI. The experiment showed that the size was correct. The DNA sequencing results are consistent with the design after DNAMan comparison, and the results show that the recombinant plasmid pET-32a-NP is successful, see attached figure 1 .
Embodiment 3
[0064] Example 3: Induced expression of prokaryotic expression vector pET-32a-NP
[0065] Based on the prokaryotic expression vector pET-32a-NP obtained in Example 2, the plasmid pET-32a-NP transformed into E.coli BL21(DE3) competent cells was cultured overnight and the positive clones were added to the ampicillin (A + ) In the medium for mass propagation overnight. Take 10 mL of the cultured bacterial solution to extract the plasmid for restriction enzyme digestion and identification. The remaining bacterial solution is cultured for 4 hours, and the IPTG inducer is added at a ratio of 1:1000, and then cultured for 4 hours. The expression products are detected by SDS-PAGE electrophoresis. figure 2 Shown.
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