A fusion protein of an anti-sulfonamide nano antibody and soybean peroxidase and application of the fusion protein
A technology of sulfonamide drugs and peroxidase, which is applied in the fields of genetic engineering and immunology, can solve the problems of complex purification process, difficult unified quality control, affecting enzyme activity and quality, and achieve the effect of easy production
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Embodiment 1
[0072] Example 1 Preparation of sulfonamide drug nanobody
[0073] The sulfonamide drug hapten was coupled with bovine serum albumin and mixed with Freund's adjuvant to immunize alpaca, and the serum titer, specificity and inhibition rate were monitored by ELISA. Peripheral blood of alpaca was collected, lymphocytes were separated by density gradient centrifugation, RNA was extracted by Trizol method and reverse transcribed into cDNA. Using cDNA as a template, the heavy chain variable region gene VHH was amplified by PCR, and phagemid pAK100-VHH was constructed after enzyme digestion and ligation. The phagemid was transformed into Escherichia coli XL1-Blue by electric shock transformation, and the helper phage M13KO7 was added at a multiplicity of infection of 20:1 in the logarithmic phase. After overnight culture, the phage was collected by the PEG-NaCl method to obtain anti-sulfonamide drugs Phage Display Nanobody Libraries. The solid-phase affinity panning method was used...
Embodiment 2
[0074] Example 2 Codon optimization and synthesis of sulfa drug nanobody and soybean peroxidase gene
[0075] According to the preferred codons of insect cells, the nanobody shown in SEQ ID NO:3 and the soybean peroxidase gene shown in SEQ ID NO:6 are codon-optimized, and the host Spodoptera frugiperda is selected, according to The preference of the host amino acid codon is optimized, and it is ensured that XbaI, NcoI and NotI are not contained in the sequence, and finally the codon-optimized Nanobody gene and soybean overexpression shown in SEQ ID NO: 2 and SEQ ID NO: 5 are obtained. The oxidase gene encodes nanobodies and soybean peroxidase with the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4, respectively.
Embodiment 3
[0076] Example 3 Construction of pVL1393-VHH-SBP expression vector
[0077] 1. Using the synthesized Nanobody gene shown in SEQ ID NO: 2 and the soybean peroxidase shown in SEQ ID NO: 5 as templates, using the following primers (primers F1, R1, F2, R2), by overlapping Extended PCR to splice the nanobody gene and soybean peroxidase gene, and introduce promoter, terminator, His tag, connecting peptide and enzyme cutting site at the same time, electrophoresis the PCR product through 1% agarose gel, and recover the target Bands to obtain spliced PCR products.
[0078] Among them, the primers are:
[0079] Primer F1: 5′-ctagtctagaatgcatcatcatcatcatcatcaagtgcagctggtggagtccggtgg-3′,
[0080] Primer R1: 5′-ggagccgccgccgccagaaccaccaccaccaccatgggcggaggacacagtcacttg-3′,
[0081] Primer F2: 5′-ggcggcggcggctccggtggtggtggttctatgggttccatgcgtctg-3′,
[0082] Primer R2: 5'-ctaggcggccgcttttcagtggtggtggtggtgatgcttggactgagcgaccagctt-3'.
[0083] Carry out three rounds of PCR reactions alto...
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