Anti-human PD-1 protein antibody as well as encoding gene and application thereof
A PD-1, encoding gene technology, applied in the field of genetic engineering, can solve the problems of poor prognosis of tumor patients, and achieve the effect of preventing and treating tumor diseases with high affinity
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Embodiment 1
[0033] Example 1 Construction of fully human single-chain antibody library for ribosome display
[0034] The DNA sequence construction process of fully human antibody libraries (T7-VH-Vκ-Cκ and T7-VH-Vλ-Cκ) for ribosome display, such as figure 2 shown.
[0035] The specific construction process is as follows:
[0036] 1. Amplify human antibody heavy chain and light chain variable region DNA
[0037] Using polyA+RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes as templates, using oligo(dT) and random primers (random primers), using reverse transcriptase kit (purchased from Clontech), poly A+ RNA was reverse transcribed into cDNA according to the method guide provided by the Clontech kit.
[0038] Using the above cDNA as a template, use a series of primers that recognize the human antibody heavy chain variable region (VH) and light chain variable region (VL) genes to perform PCR amplification to obtai...
Embodiment 2
[0103] Example 2 Ribosome display screening of fully human single-chain antibodies that bind to PD-1 protein
[0104] The process of screening single-chain antibodies by ribosome display is as follows: image 3 shown. Because the scFv single-chain antibody library DNA prepared in Example 1 above does not contain a termination code, it is transcribed and translated in vitro to produce a complex formed by Antibody-Ribosome-mRNA ("ARM complex ").
[0105] Antibodies on the ARM complex were screened with antigen immobilized on the solid phase. After the non-specific ARM complex is eluted, RT-PCR method is used to obtain the DNA capable of binding the antigen to the antibody. Re-add T7 promoter etc. by PCR method to regenerate and obtain full-length DNA that can be used for ribosome display.
[0106] (1) Biotin-coupled PD-1 protein to avidin magnetic beads
[0107] Human PD-1 protein (catalog number 10377-H03H) was purchased from Beijing Sino Biological Technology Co., Ltd.; E...
Embodiment 3
[0142] Example 3 Single-chain antibody expression and purification
[0143] The gene encoding the single-chain antibody #hPD1-B was cloned into the expression vector pET27b(+), and pET27b-HPD1b was constructed;
[0144] Transform pET27b-HPD1b into expression bacteria E.coli BL21(DE3), and induce expression with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N-terminal of the scFv is the pelB sequence, and the pelB sequence can be The expressed scFv is secreted into the periplasmic space of BL21(DE3); the C-terminus of the scFv contains an HSV tag and a 6×His tag to facilitate the purification of the target protein;
[0145] The method provided by Qiagen was conveniently separated and purified with Ni-NTA column to obtain the single-chain antibody against human PD-1 protein.
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