Specific antibody of pepsinogen I as well as preparation method and application thereof
A pepsinogen and specific technology, applied in the biological field, can solve the problems of unfavorable long-distance transportation and popularization of detection, large-scale mass production of high finished products, unfavorable large-scale mass production, etc. The effect of modernized production and simple structure
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Embodiment 1
[0095] Example 1: Preparation of antigens
[0096] The DNA sequence encoding pepsinogen I was constructed into pFASTBac-HTA expression vector, and PGI with histidine tag (6*His-tag) was recombinantly expressed in SF9 insect cells, and then extracted and purified as antigen.
[0097]所述抗原如有如SEQ ID NO:1所示的氨基酸序列,具体为:MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNPARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGSSNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEGSCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA
Embodiment 2
[0098] Example 2: Alpaca immunization injection
[0099] In this example, alpacas were immunized with the antigen prepared in Example 1. Specific steps are as follows:
[0100] (1) The antigen prepared in Example 1 was equally divided into 4 parts, each of about 0.25 mg;
[0101] (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.
[0102] 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 periphe...
Embodiment 3
[0103] Example 3: Construction of Antibody Libraries
[0104] 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:
[0105] (1) Using density gradient centrifugation to separate lymphocytes from the peripheral blood of alpaca veins;
[0106] (2) Extracting the total mRNA of lymphocytes and reverse transcribing into cDNA;
[0107] (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;
[0108] (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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