CN108251380A - A kind of 7E8 cell strain of monoclonal antibody preparation method of resistant to foot and mouth disease 3B albumen and application - Google Patents

Abstract

7E8 cell strain of monoclonal antibody preparation method and application the invention discloses a kind of resistant to foot and mouth disease 3B albumen, the bacterial strain on October 25th, 2017, are preserved in China typical culture collection center, deposit number is CCTCC NO:C2017228.The present invention can be with the content of 3B albumen in qualitative detection serum, whether so as to infer mouth disease virus infection.Mainly for detection of foot and mouth disease virus non-structural protein 3B antigens in animal blood serum, monitor and assess suitable for animal population infection state, find early stage subclinical infection and quarantine of passing in and out, do not limited by serotype.

Description

A kind of 7E8 cell strain of monoclonal antibody preparation method of resistant to foot and mouth disease 3B albumen and Using

Technical field

The invention belongs to biotechnology, specifically, being related to a kind of 7E8 monoclonal antibodies of resistant to foot and mouth disease 3B albumen Cell strain preparation method and application.

Background technology

FMD is the artiodactyls deadly infectious disease as caused by FMDV, and World Organization for Animal Health (OIE) is included in must The animal epidemic that must be notified to.Disease propagation is fast, host range is wide, incidence is high, and great threat is formed to animal husbandry development.In recent years Carry out China's livestock breeding industry to quickly grow, aftosa is great potential threat.Policy is slaughtered with what developed country was usually taken Difference, developing country generally control the disease using immune method.Therefore, how immune animal is quickly and reliably distinguished And infection animal, it is particularly important for the anti-system of FMD and international trade.After zoogenetic infection FMDV, structure can be generated Protein antibodies, and NSP antibody can be generated, and vaccine immunity animal mainly induces body to generate FMDV structural proteins antibody, with knot Structure albumen is compared, and the variation of non-structural protein is less, relatively conservative.In FMD diagnosis, distinguish immune animal and natural infection is moved Object is a highly important content, differentiates that natural infected animal and immune animal are also that OIE judges a country whether there is FMD Popular foundation.In recent years, the diagnostic method of detection FMDVNSP antibody is established successively, to distinguish FMDV infection animals and note Penetrate vaccine animal.

The NSP of FMDV refers to all by virus other than capsid protein 1A, 1B, 1C, 1D and their precursor protein The albumen of viral capsid composition is encoded but is not involved in, before maturation protein L, 2A, 2B, 2C, 3A, 3B, 3C, 3D and they Body.The 3B albumen of FMDV is by the connected but not exactly the same gene code in non-structural protein P3 code areas 3, size difference For 69bp, 72bp, 72bp and 3 kinds of different 3B albumen (VPg1~3) are generated, this phenomenon is rare in RNA virus.VPg albumen Participate in the starting synthesis of RNA virus and the formation of capsid.Since V Pg carry pUpU structures and newly synthesized filial generation RNA phases Even, so VPg is considered as the primer of picornavirus R NA synthesis.This special rna replicon mode is transcribed with host mRNA It is different.Thus, when host RN A synthesis is suppressed, have no effect on the synthesis of FMDVRNA.The copy number and RNA of VPg genes Virus appeal be proportionate, VPg mutation viral lifecycle will be caused to extend, this also means that the synthesis of polymeric protein and Processing is postponed, and infectious number of particles is reduced.At present, the research about FDMV3B albumen is less, it is in virus replication In effect and disclosed completely not yet with the interaction of host.

Invention content

The purpose of the present invention is to provide a kind of 7E8 cell strain of monoclonal antibody preparation methods of resistant to foot and mouth disease 3B albumen And application, to express albumen 3B as mice immunized with antigen, by hybridoma technology obtain specific hybridoma cell strain and Its monoclonal antibody, establishes the method for competitive ELISA detection method detection 3B albumen, and assembles kit, to foot and mouth disease virus Infection animal carries out antidiastole.

Its specific technical solution is：

A kind of 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen, the bacterial strain on October 25th, 2017, are protected China typical culture collection center is hidden in, deposit number is CCTCC NO:C2017228.Depositary institution address：Hubei Province is military Han Shi Wuchang Districts Luo Jia Shan (Bayi Road 299).

Further, the nucleotide sequence of the bacterial strain such as SEQ:ID:NO:Shown in 1.

GAAGGACCCTACACCGGTCCACTCGAGCGTCAAAAACCTCTGAAAGTGAGAGCCAAGCTCCCACAGCAGGAGGGG CCCTACGCTGGTCCGATGGAGAGACAGAAACCGCTGAAAGTGAAAGTGAAAGCCCCGGTCGTTAAGGAAGGACCTTA CG AAGGACCGGTGAAGAAACCTGTCGCTTTGAAAGTGAAAGCAAAGAACTTGATTGTCACTGAG

A kind of preparation method of the 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen, includes the following steps：

Step 1,3B protein immunization mouse；

Step 2 takes spleen and SP2/0 cells to carry out cell fusion；

Step 3, the cell strain of ELISA screening specificity and by positive hole cell monoclonal；

It is prepared by step 4, ascites；

Step 5, Protein G affinity purifications are monoclonal antibody-purified.

The 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen of the present invention is preparing detection aftosa 3B albumen examinations The application of agent box process.

Compared with prior art, beneficial effects of the present invention are：

The present invention can be with the content of 3B albumen in qualitative detection serum, whether so as to infer mouth disease virus infection.Mainly For detecting foot and mouth disease virus non-structural protein 3B antigens in animal blood serum, monitor and comment suitable for animal population infection state Estimate, find early stage subclinical infection and quarantine of passing in and out, do not limited by serotype.

Description of the drawings

Fig. 1 is 3B expression proteantigen SDS-PAGE electrophoresis；

Fig. 2 is monoclonal antibody SDS-PAGE electrophoresis.

Specific embodiment

Technical scheme of the present invention is described in more detail with reference to specific embodiment.

A kind of preparation method of the 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen, which is characterized in that including following Step：

Step 1: immune programme

Table 1

Step 2 exempts from rear mice serum bioactivity

Table 2

Note：

Coating condition：10 μ g/ml envelope antigens, 100 μ l/well, 37 DEG C of 150 μ l/ of water-bath coating 2.5h → confining liquid Well37 DEG C of water-bath 2h；

ELISA reaction conditions：100 μ l/well of primary antibody, 37 DEG C of 100 μ l/well of water-bath 1.5h → sheep anti-mouse igg-HRP 37 DEG C of 100 μ l/well color development at room temperature 15min → 1N hydrochloric acid of water-bath 1.5h → TMB, 50 μ l/well terminations → microplate reader OD450 is read Value.

Step 3, fusion mice serum bioactivity

Table 3

Note：

Coating condition：10 μ g/ml envelope antigens, 100 μ l/well, 37 DEG C of 150 μ l/ of water-bath coating 2.5h → confining liquid Well37 DEG C of water-bath 2h；

Reaction condition：50 μ l/well of primary antibody, 37 DEG C of water-bath 45min → sheep anti-mouse igg-HRP 50 μ l/well, 37 DEG C of water Bathe 50 μ l/well color development at room temperature 10min → 1N hydrochloric acid of 45min → TMB, 50 μ l/well terminations → microplate reader OD450 readings.

Step 4, cell fusion

By the mouse boosting cell being immunized and SP2/0 myeloma cell with 5~7：1 ratio is merged with 5% PEG6000 Mouse boosting cell from the mouse spleen being immunized is taken out and is added dropwise in SP2/0 myeloma cell, side edged mixes rhythm, Later, it is stored at room temperature 10 minutes.And by the cell seeding after fusion in containing thymus gland sertoli cell, HAT, 20% calf serum, In the semisolid culturemedium of Methyl-Cellulose methylcellulose and ISCOVE culture solutions.In 37 DEG C, 5%CO₂Incubator is trained After supporting 7-10 days, it is seen that there are a large amount of hybridoma clones to occur in semisolid culturemedium.It is provoked gram with sterile water dropper under the microscope It is grand and plant in being covered with thymus gland sertoli cell as feeder cells, the 96 hole cells of HAT, 20% calf serum and ISCOVE culture solutions In culture plate, with infinite dilution method, a clone i.e. detectable supernatant antibody titer after a week is only cultivated per hole.

The screening of step 5, anti-3B antibody-secretings positive colony

The clone of the anti-3B antibody-secretings positive is screened using ELISA method.

Step 6, ascites prepare with it is monoclonal antibody-purified.

There was only heavy chain and light chain after monoclonal antibody SDS-PAGE after purification as can be seen from Figure 2.

The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention is without being limited thereto, it is any ripe Those skilled in the art are known in the technical scope of present disclosure, the simple of technical solution can be become apparent to Variation or equivalence replacement are each fallen in protection scope of the present invention.

Sequence table

<110>Xinjiang herding academy of sciences veterinary institute

<120>A kind of 7E8 cell strain of monoclonal antibody preparation method of resistant to foot and mouth disease 3B albumen and application

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<170> SIPOSequenceListing 1.0

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<211> 216

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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gaaggaccct acaccggtcc actcgagcgt caaaaacctc tgaaagtgag agccaagctc 60

ccacagcagg aggggcccta cgctggtccg atggagagac agaaaccgct gaaagtgaaa 120

gtgaaagccc cggtcgttaa ggaaggacct tacgaaggac cggtgaagaa acctgtcgct 180

ttgaaagtga aagcaaagaa cttgattgtc actgag 216

Claims (4)

1. a kind of 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen, which is characterized in that the bacterial strain is in 2017 10 The moon 25, China typical culture collection center is preserved in, deposit number is CCTCC NO:C2017228.

2. the 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen according to claim 1, which is characterized in that described The nucleotide sequence of bacterial strain such as SEQ:ID:NO:Shown in 1.

3. a kind of preparation method of the 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen, which is characterized in that including following step Suddenly：Step 1,3B protein immunization mouse；

Step 2 takes spleen and SP2/0 cells to carry out cell fusion；

Step 3, the cell strain of ELISA screening specificity and by positive hole cell monoclonal；

It is prepared by step 4, ascites；

Step 5, Protein G affinity purifications are monoclonal antibody-purified.

4. the 7E8 cell strain of monoclonal antibody of resistant to foot and mouth disease 3B albumen described in claim 1 is preparing detection aftosa 3B albumen The application of kit process.