Specific antibody of novel coronavirus N protein as well as preparation method and application of specific antibody
A specific and antibody technology, applied in the field of specific antibodies of the new coronavirus N protein and its preparation, can solve problems such as unfavorable long-distance transportation and universal testing, large-scale mass production of finished products, and unfavorable large-scale mass production. Low-temperature storage and transportation, large-scale production, and simple structure
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Embodiment 1
[0094] Example 1: Preparation of antigens
[0095] The DNA sequence encoding the N protein RBD region was constructed into the Pet-28a expression vector, and the N protein RBD domain with histidine tag (6*His-tag) was recombinantly expressed in E. coli, and then extracted and purified as an antigen.
[0096] The antigen has the amino acid sequence shown in SEQ ID NO: 1, specifically: NNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEG
Embodiment 2
[0097] Example 2: Alpaca immunization injection
[0098] In this example, alpacas were immunized with the antigen prepared in Example 1. Specific steps are as follows:
[0099](1) The antigen prepared in Example 1 was equally divided into 4 parts, each of about 0.25 mg;
[0100] (2) The alpacas were immunized 4 times in total, and the antigens were subcutaneously injected into the animals. The first immunization was recorded as the first day, and the subsequent immunizations were on the 10th, 19th, and 28th days respectively; On the 28th day, before the fourth immunization injection, about 200 mL of alpaca venous peripheral blood was collected, and on the 42nd day, that is, 14 days after the fourth immunization, about 300 mL of alpaca venous peripheral blood was collected.
[0101] Compared with the traditional immunization technical solutions for animal antibodies such as mice and rabbits, the method provided in this example collects a large amount of alpaca venous peripher...
Embodiment 3
[0102] Example 3: Construction of Antibody Libraries
[0103] Using the two batches of alpaca venous peripheral blood collected in Example 2 as raw materials, a high diversity Nanobody library was constructed. The two batches of alpaca venous peripheral blood were treated in the same way, specifically:
[0104] (1) Using density gradient centrifugation to separate lymphocytes from the peripheral blood of alpaca veins;
[0105] (2) Extracting the total mRNA of lymphocytes and reverse transcribing into cDNA;
[0106] (3) Using appropriate DNA primers, the above-mentioned cDNA is used as a template, and the VHH fragments of alpaca immunoglobulin IgG2 and IgG3 are obtained by polymerase chain reaction (PCR) amplification, that is, the DNA fragments of Nanobodies;
[0107] (4) connecting the DNA of VHH to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein, pIII is a protein present on the phage surface flagella;...
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