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Porcine-derived osteopontin (OPN) monoclonal antibody, hybridoma cell strain thereof, and applications of monoclonal antibody and hybridoma cell strain

A technology of hybridoma cell line and monoclonal antibody, which is applied in the field of bioengineering, can solve the problems of not single identified parts, false positives, deep background, etc., and achieve the effect of high application value

Active Publication Date: 2020-05-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the porcine OPN polyclonal antibody can recognize multiple epitopes, and there are many non-specific bands when doing western blot; when doing immunohistochemistry, the background is darker, and the probability of false positives is relatively high; when doing immunofluorescence, the identified Not a single part

Method used

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  • Porcine-derived osteopontin (OPN) monoclonal antibody, hybridoma cell strain thereof, and applications of monoclonal antibody and hybridoma cell strain
  • Porcine-derived osteopontin (OPN) monoclonal antibody, hybridoma cell strain thereof, and applications of monoclonal antibody and hybridoma cell strain
  • Porcine-derived osteopontin (OPN) monoclonal antibody, hybridoma cell strain thereof, and applications of monoclonal antibody and hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: In vitro acquisition of porcine OPN protein

[0034] Referring to Examples 1 and 2 in Chinese patent application 201811476996.8 "A Recombinant Lentiviral Vector, Recombinant Lentivirus and Its Application", the porcine OPN recombinant protein was obtained. Specifically, it includes: predicting the signal peptide of the OPN protein through the signal peptide prediction software SignalP4.1Server, removing its own signal peptide, and adding a 6'His sequence in front of the gene sequence, that is, the final sequence is a 6'His-OPN sequence. Ligate the target sequence into the lentiviral modified vector pCDH-CMV-HSA-MCS-6His-EF1-GFP+Puro to obtain the recombinant lentiviral vector pCDH-CMV-HSA-6′His-OPN-6His-EF1-GFP+Puro . Through the lentiviral packaging system, the packaging plasmids (pMD2.G and psPAX2) and the lentiviral recombinant vector were co-transfected into 293FT cells to obtain the virus containing the target gene. The virus was infected into the CHO-...

Embodiment 2

[0035] Example 2: Preparation of porcine OPN mouse monoclonal antibody

[0036] The purified porcine OPN recombinant protein expressed in the eukaryotic system in Example 1 was used as an immunogen to immunize mice. Select purebred BABL / c female mice of SPF grade for 6-8 weeks to immunize with porcine OPN recombinant protein. For the first immunization, 100 μg of porcine OPN recombinant protein plus Freund’s complete adjuvant (mixed in equal volumes) was subcutaneously injected into multiple points; at an interval of three weeks, the second immunization of 100 μg of porcine OPN recombinant protein was subcutaneously injected into multiple points with Freund’s incomplete adjuvant. The total volume is 200 μL; at an interval of three weeks, the third immunization dose is halved, that is, 50 μg of protein without adjuvant is injected intraperitoneally, blood is collected from the orbit 7 days later, and the titer is measured by ELISA. After the titer reaches a certain level, the b...

Embodiment 3

[0067] Example 3 Monoclonal Antibody Purification

[0068] 1. Caprylic acid-ammonium sulfate precipitation crude extraction

[0069] Add 2 parts of 0.06mol / L pH5.0 acetate buffer to 1 part of centrifuged pretreated ascites, adjust the pH to 4.8 with 1mol / L HCl solution; Add octanoic acid, finish adding within 30min, let stand at 4°C for 2h, centrifuge at 2000rpm for 30min, discard the precipitate; filter the supernatant through a nylon sieve (125μm), add 1 / 10 volume of 0.01mol / L PBS buffer, and use 1mol / L Adjust the pH to 7.2 with NaOH; add saturated ammonium sulfate to 45% saturation at 4°C for 30 minutes, let stand for 1 hour; centrifuge at 12000rpm for 30 minutes, discard the supernatant; dissolve the precipitate in an appropriate amount of PBS buffer (containing 137mmol / L NaCl, 2.6mol / L KCl, 0.2mmol / L EDTA (ethylenediaminetetraacetic acid)), dialyze in 50-100 times volume of PBS buffer, overnight at 4°C, change water more than 3 times during the period; centrifuge at 120...

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Abstract

The invention discloses a porcine-derived osteopontin (OPN) monoclonal antibody, a hybridoma cell strain thereof, and applications of the monoclonal antibody and the hybridoma cell strain. The hybridoma cell strain secreting the porcine-derived OPN monoclonal antibody is classified and named as a hybridoma cell strain Cy2, and is preserved in China Center for Type Culture Collection (CCTCC) on Dec.19,2019, wherein the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO:C2019293.The porcine-derived OPN monoclonal antibody is secreted by the hybridomacell strain or a passage cell strain of the hybridoma cell strain. The monoclonal antibody is suitable for rapidly and accurately detecting the content of OPN protein in pig tissues, semen or pig oocytes, so that the monoclonal antibody can be used for preparing related detection kits or colloidal gold test paper, and a firm foundation is laid for deep research of porcine-derived OPN.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a porcine OPN monoclonal antibody, its hybridoma cell line and application. Background technique [0002] With the African swine fever, the pig industry has been hit hard. Although the situation is under control, the pig raising market is not optimistic. How to restore the production performance as soon as possible and how to improve the reproductive performance of boars? Farms use technical means to select boars with high fertility for semen collection and mating, but collecting data related to fertility is time-consuming and laborious (Leno-Colorado et al, 2017). The best way is to use new technology to assist breeding. Porcine seminal plasma and sperm membrane proteins have been implicated in sperm maturation and protection mechanisms (Dietrich et al, 2019; Druart et al, 2019). These proteins can improve the quality of sperm, and through screening, other proteins can...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/24G01N33/68G01N33/577
CPCC07K16/24G01N33/577G01N33/6863G01N2333/52
Inventor 张守全陈云王凯赵志宏
Owner SOUTH CHINA AGRI UNIV
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