Hybridoma cell line and preparation method thereof, monoclonal antibodies, application and kit

A hybridoma cell line, monoclonal antibody technology, applied in the fields of preparation, hybridoma cell line, monoclonal antibody, application and kits, can solve the problems of unknown mechanism of action and functional application, shorten the empty period and save costs , the effect of significant economic benefits

Active Publication Date: 2019-05-17
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, some scholars have further studied and found that Hp protein is secreted in both pregnant and non-pregnant endometrial tissues, but the content is the most abundant in the decidua tissue, followed by the secretory endometrium, and the lowes...

Method used

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  • Hybridoma cell line and preparation method thereof, monoclonal antibodies, application and kit
  • Hybridoma cell line and preparation method thereof, monoclonal antibodies, application and kit
  • Hybridoma cell line and preparation method thereof, monoclonal antibodies, application and kit

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Recombinant baculovirus rBV-hp infects sf9 cells to express Hp

[0036] 1. Design of primers

[0037]According to the Hp gene sequence published on GeneBank (NM_001040470), a pair of specific primers F1 / F2 for amplifying the Hp gene was designed, and an NcoI restriction site was inserted in the upstream primer, and a HindIII restriction site was inserted in the downstream primer. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd. Primers are as follows:

[0038] F1: AAA CCATGG CGATGAGCGCCCTGCAA NCOI

[0039] F2: TTT AAGCTT TTAGTTGTTAGCGATGG HindIII

[0040] 2. PCR amplification

[0041] The HP gene fragment was amplified using the bovine blood RNA of pregnant dairy cows isolated in our laboratory as a template. 35 cycles; ③72°C for 10 minutes. After the reaction, it was detected by 0.8% agarose gel electrophoresis.

[0042] 3. Construction of recombinant transfer plasmid pFastBacHTB-Hp

[0043] The PCR positive product wa...

Embodiment 2

[0054] Embodiment 2: Preparation of monoclonal antibody

[0055] 1) Preparation of monoclonal antibody

[0056] After the Hp protein expressed by the recombinant baculovirus was purified through the column, the protein concentration was calculated according to the formula. BALB / C mice were immunized with the Hp protein as an antigen.

[0057] Purified Hp protein was used as an immune antigen to immunize 6-week-old female BABLB / C mice five times with an interval of 2-3 weeks. For the first immunization, Hp protein was added with the same amount of Freund's complete adjuvant, and the immunization dose was 100 μg per mouse. The immunization route was subcutaneous injection at multiple points on the back of the neck, and Freund's incomplete adjuvant was used later. 12-15 days after the fourth immunization, a small amount of blood was collected from the tail, and the serum titer was determined by indirect ELISA. If the serum titer was qualified, booster immunization was perfo...

Embodiment 3

[0075] Embodiment 3: the hybridoma cell line that secretes the monoclonal antibody of anti-Hp protein

[0076] The embodiment of the present invention discloses a hybridoma cell line secreting a monoclonal antibody against Hp protein: the hybridoma cell line is the hybridoma cell line of CCTCC NO: C2017119 (i.e. hybridoma cell line 4F 11 ) and the hybridoma cell line of CCTCC NO: C201858 (that is, the hybridoma cell line 12F 9 ).

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Abstract

The invention provides a hybridoma cell line and a preparation method thereof, monoclonal antibodies, an application and a kit, and relates to the technical field of animal genetic engineering. The hybridoma cell line secreting the anti-Hp protein monoclonal antibodies is a combination of a hybridoma cell line of CCTCC NO:C2017119 and a hybridoma cell line of CCCC NO:C201858. The invention also discloses the anti-Hp protein monoclonal antibodies. The monoclonal antibodies are selected from monoclonal antibodies secreted by the matched hybridoma cell line of CCTCCNO: C2017119 and monoclonal antibodies secreted by the matched hybridoma cell line of CCCCNO:C201858. The antibodies secreted by the two hybridoma cell lines are used for preparing the ELISA kit for early pregnancy detection in dairy cows. The ELISA kit has good sensitivity and high specificity, and facilitates earlier detection of early pregnancy in cattle herd.

Description

technical field [0001] The invention relates to the technical field of animal genetic engineering, in particular to a hybridoma cell line, a preparation method, a monoclonal antibody, an application and a kit. Background technique [0002] Currently, rectal examination and ultrasonography are the most widely used diagnostic methods in clinical practice. However, the rectal examination is generally carried out 60 days after the cow is mated, and some even take longer to make a diagnosis, which not only affects the reproductive cycle of the cow, but also affects the performance of the cow's production performance. Ultrasonic testing is expensive and susceptible to infection. The bovine pregnancy-associated protein (PAG) ELISA kit, which is used in some cattle farms but has not been widely promoted, can make a more reliable judgment at 28 days after pregnancy. But these methods can only make an accurate diagnosis three weeks after artificial insemination of cows. However, st...

Claims

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Application Information

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IPC IPC(8): C12N5/20C12N15/866C07K16/18G01N33/577G01N33/68
Inventor 向敏程蕾胡修忠周源余婕刘晓华王定发夏瑜陶弼菲
Owner WUHAN ACADEMY OF AGRI SCI
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