Porcine reproductive and respiratory syndrome virus antibody detection method and application thereof
A respiratory syndrome and antibody detection technology, applied in measuring devices, disease diagnosis, biological testing, etc., can solve problems such as non-production, undetectable antibodies, false negatives, etc., to save cumbersome processes and make up for deficiencies and defects Effect
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Embodiment 1
[0034] Example 1 Acquisition of NSP4 Gene
[0035] A. RNA extraction: 200 μL of HP-PRRSV HuN4 vaccine (purchased from Harbin Veken Biotechnology Co., Ltd.) was added with the same volume of Trizol, PRRSV RNA was extracted according to the instructions, the RNA concentration was measured, and then put in a -80°C refrigerator for later use.
[0036] B. Reverse transcription: reverse transcription of the extracted RNA, the reverse transcription system is 20 μL, including: 5× reverse transcriptase Buffer 2 μL, 10mM dNTP MiX 4 μL, RNase inhibitor 1 μL, oligo(dT)18primer 1 μL, AMV Reverse transcriptase 1 μL, RNase-free water 1 μL, template RNA 1 μg; reaction conditions were 42°C, 60 min; 70°C, 5 min.
[0037]C. PCR amplification of the target fragment: use the cDNA synthesized by reverse transcription as a template for PCR amplification. The reaction system was 50 μL: 1 μL each of the upstream and downstream primers, 25 μL of TaKaRa Premix Taq, 2 μL of cDNA, and water to 50 μL. Re...
Embodiment 2
[0043] Embodiment 2 NSP4 expression and purification
[0044] A. Expression and identification of NSP4 protein: Transform the recombinant expression plasmid pET28a-NSP4 into the competent cells of the expression strain Trasseta (DE3), use the kana resistance to screen the recombinant transformants, pick a single colony and activate it in LB medium overnight. The ratio of 1% was transferred to fresh LB medium containing kana-resistant and cultured until the OD600 was about 0.8, adding IPTG with a final concentration of 1 mM, 225 rpm, 30°C shaking culture for 5 hours. The cells were collected by centrifugation at 10,000 g at 4°C for 10 min, resuspended in PBS, ultrasonically disrupted, and centrifuged at 12,000 rpm at 4°C for 15 min, and the supernatant and precipitate were collected for SDS-PAGE identification. The results showed that the target protein was highly expressed, with a size of about 26KDa, and most of the target protein was located in the lysed supernatant.
[004...
Embodiment 3E
[0047] Embodiment 3ELISA condition optimization
[0048] Each step in the ELISA process is optimized, and the optimized result is:
[0049] A. Antigen coating amount: 50-1000ng of purified NSP4 was used as the coating protein, and it was found that the P / N value was the highest when coating 200ng;
[0050] B. Coating conditions: Different coating buffers and action time were detected, and it was found that the coating buffer was a phosphate buffer with pH=7.2, and its 1L solution contained 0.356g NaH 2 PO 4 , 2.772 g Na 2 HPO 4 12H 2 O, 8.5g NaCl, the highest P / N value when coated overnight;
[0051] C. Blocking conditions: with the blocking solution containing 2.5% skimmed milk powder with a pH of 7.2 PBS'T buffer, the P / N value is the highest when incubated at 37°C for 1 hour;
[0052] D. Serum sample dilution: the PBS'T+volume concentration that is 7.2 is 2.5% skimmed milk powder and is diluted by 1:40, and the P / N value is the highest when adding 100 μ l;
[0053] E...
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