Nano-antibody and bivalent neutralizing nano-antibody for resisting porcine circovirus type 2 Cap protein and application thereof
A technology of porcine circovirus and nano-antibody, applied in anti-viral immunoglobulin, anti-viral agent, application, etc., can solve the problems of restricting the development and application of bispecific whole-molecular antibodies, complex structure, and many disulfide bonds, and achieve Effective immune protection, high application value effect
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Embodiment 2
[0066] Example 2 Construction of Anti-PCV2 Nanobody Immune Library
[0067] 2.1 Lymphocyte isolation and total RNA extraction
[0068] Mononuclear lymphocytes were isolated from alpaca peripheral blood by density gradient centrifugation according to routine procedures in the technical field. Lymphocytes were counted using a hemocytometer, and 1.0×10 7 Total RNA was extracted from living cells, and the remaining lymphocytes were frozen at -80°C.
[0069] From 1.0×10 according to routine procedures in this technical field 7 Total RNA was extracted from lymphocytes, and the concentration was determined by NanoDrop2000, which was then used for reverse transcription to synthesize first-strand cDNA.
[0070] 2.2 Synthesis of cDNA by reverse transcription
[0071] According to the instructions of the first-strand cDNA synthesis kit (ThermoScientificRevertAidFirstStrandcDNASynthesisKit, K1621), the total RNA obtained in step 2.1 was used as a template to perform reverse transcript...
Embodiment 3
[0087] Example 3 Screening and Identification of Anti-PCV2 Cap-specific Nanobodies
[0088] 3.1 Phage amplification
[0089] The primary antibody library was formed after the ligation product of VHH and pCANTAB5E was electrotransformed into E.coliTG1 (step 1.6); take 1 branch (1 mL) of the primary antibody library and add it to 250 mL of 2×YT-G (2% glucose) medium , 37°C, 250rpm, cultured for 1h to OD600 of about 0.5, added helper phage M13KO7 with a final concentration of 100μg / mL Amp and Moi=20:1, gently shaken in a water bath at 37°C for 30min, and then cultivated on a shaker at 37°C for 30min; centrifuged at 4000rpm After discarding the supernatant, use 2 times the volume (500mL) of 2×YT-AK medium to suspend the bacterial pellet, culture overnight at 37°C, 220rpm; centrifuge to take the supernatant, add 100mL of PEG / NaCl (PEG is the product of Aladdin, CAS 25322-68-3; NaCl is a product of Shanghai Sinopharm, CAS 7647-14-5), in an ice bath at 4°C for no more than 1 hour. ...
Embodiment 4
[0104] Example 4 Differential Analysis of Anti-PCV2 Nanobodies from 3 Different Antigen Immune Libraries
[0105] Using PCV2-VLP recombinant protein as an antigen, the immune libraries from PCV2 whole virus inactivated vaccine, PCV2-VLP vaccine and PCV2 genetically engineered subunit vaccine were screened, and a total of 11 independent anti-PCV2 nanobody sequences were obtained . Among them, 4 kinds of anti-PCV2 nanobodies (Nbvr-4, Nbvr-20, Nbvr-23, Nbvr-67) with different sequences were obtained by panning from the PCV2 whole virus inactivated vaccine immune library, and obtained from the PCV2-VLP vaccine immune library Five independent anti-PCV2 nanobody sequences (Nbvl-11, Nbvl-18, Nbvl-22, Nbvl-55, Nbvl-56) were obtained by panning, and two were obtained by panning from the PCV2 genetic engineering subunit vaccine immune library. Independent anti-PCV2 Nanobody sequences (Nbcp34, Nbcp71).
[0106] The 11 kinds of anti-PCV2 nanobody sequence coding genes were amplified and...
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