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Clone, expression and application of BALF4 polypeptide

An expression vector and expression method technology, applied in the field of molecular biology, can solve the problem of poor immunogenicity of anti-EB virus vaccine, and achieve the effect of strong immunogenicity

Inactive Publication Date: 2017-09-15
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The first object of the present invention is to provide a nucleotide sequence encoding BALF4 polypeptide, the second object of the present invention is to provide a recombinant expression vector, the third object of the present invention is to provide recombinant cells, and the fourth object of the present invention To provide the BALF4 polypeptide, the fifth purpose of the present invention is to provide a method for expressing the BALF4 polypeptide, and the sixth purpose of the present invention is to provide the application of the BALF4 polypeptide to alleviate the technical problem of poor immunogenicity of the anti-Epstein-Barr virus vaccine in the prior art

Method used

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  • Clone, expression and application of BALF4 polypeptide
  • Clone, expression and application of BALF4 polypeptide
  • Clone, expression and application of BALF4 polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Expression and purification of embodiment 1 recombinant protein

[0042] 1. Reagents

[0043] Escherichia coli DH5α: Cultivate overnight in 5ml LB containing 50-100mg / ml kanamycin, take 0.85ml of culture solution and add 0.15ml of sterilized glycerol to mix completely, transfer to a cryopreservation bottle, freeze in liquid nitrogen or dry ice / alcohol bath at -80°C.

[0044] Pichia pastoris recombinant expression vector pPICZαA: add His at the 5' end of the sequence shown in SEQ ID NO.1 6 Label, add a stop codon at the 3' end, introduce EcoR I and Xba I restriction sites at the 5' end and 3' end respectively; α-factor secretion signal secretes and expresses the target protein. When expressed, the gene of interest is inserted into the reading frame of the start codon of the signal sequence.

[0045] Pichia pastoris GS115 strain was purchased from Takara.

[0046] RPMI-l640 medium, reverse transcription kit, PCR reagent, restriction endonuclease, T4 ligase, plasmid e...

Embodiment 2

[0099] Example 2 Detection of immunostimulatory activity of recombinantly expressed protein

[0100] 1. Reagents

[0101] ELISA coating buffer (pH 9.6, 1L): dissolve 1.59g Na 2 CO 3 and 2.93g NaHCO 3 In 1L double distilled water.

[0102] ELISA wash buffer (pH 7.4, 1L): Weigh 0.2g of solid KH 2 PO 4 , 2.9 g Na 2 HPO 4 12H 2 O, 8gNaCl and 0.2g KCl were dissolved in 1L of double distilled water, then 0.5ml of Tween 20 was added, mixed well and set aside.

[0103] 2. Experiment

[0104] 1. Mouse immunization experiment

[0105] BALB / c mice (female, 4-6 weeks old) were purchased from Qingdao Food and Drug Inspection Institute, and raised in the Animal Center of Qingdao University in accordance with the Animal Management Regulations of Shandong Province, and the animal laboratory was SPF grade. The mice were divided into three groups, 10 in each group. The first group was the control group, and each mouse was subcutaneously injected with 100 μl of PBS once every two week...

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Abstract

The invention provides clone, expression and application of BALF4 polypeptide and relates to the technical field of molecular biology. According to the clone, the expression and the application, through gene optimization, an optimized nucleotide sequence encoding the BALF4 polypeptide is obtained, high-expression yeast cells for recombining the BALF4 polypeptide are constructed, a purification method is optimized, and the BALF4 polypeptide is obtained finally. The BALF4 polypeptide provided by the invention can stimulate production of a high-level antigen specific antibody, induces mouse splenic lymphocyte proliferative response, can effectively stimulate a body immune system, has stronger immunogenicity, can be used as an immune adjuvant and is used for improving the prevention effect on an EB virus vaccine so as to reach the purpose of effectively preventing EB virus infection.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the cloning, expression and application of a BALF4 polypeptide. Background technique [0002] Epstein-Barr virus (EBV) belongs to the fourth type of human herpes virus and was discovered by Epstein and Barr in 1964 when they were studying malignant lymphoma in African children. Epstein-Barr virus is a lymphotropic virus, which is one of the most commonly infected viruses in humans, infecting more than 90% of the world's population. Because of its high infection rate and carcinogenicity, Epstein-Barr virus was designated as a Class I carcinogen by the International Center for Cancer Research in 1997. [0003] Epstein-Barr virus is transmitted through saliva or close oral contact. Epstein-Barr virus mainly infects B cells and epithelial cells, and the oropharyngeal epithelial cells are the primary site of Epstein-Barr virus infection and replication. Epstein-Barr virus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/05C12N15/38C12N15/81C12N1/19A61K39/39A61K39/245A61P37/04A61P31/22A61P35/00C12R1/84
CPCA61K39/12A61K39/39A61K2039/55516A61K2039/572A61K2039/575C07K14/005C12N15/815C12N2710/16222C12N2710/16234A61K2300/00
Inventor 李培峰王曼王昆吴薇张银凤
Owner QINGDAO UNIV
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