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A method for recombinantly expressing pcv2

A technology for recombinant proteins and amino acids, applied in the biological field, can solve problems such as low yield and complexity

Active Publication Date: 2021-05-28
BIORTUS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the expression of PCV2 protein through the traditional CsCl 2 The purification method of density gradient centrifugation is more complicated, and the yield is low, only about 5mg / L

Method used

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  • A method for recombinantly expressing pcv2
  • A method for recombinantly expressing pcv2
  • A method for recombinantly expressing pcv2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The main steps include: synthesizing a recombinant plasmid carrying recombinant PCV2 inserted into the His affinity tag, transforming the recombinant plasmid into DH10Bac Escherichia coli competent cells, obtaining recombinant baculovirus DNA (recombinant Bacmid) through resistance and blue-white screening, and extracting recombinant Baculovirus DNA was transfected into Sf9 insect cells to obtain recombinant baculovirus.

[0022] (1) Construction and screening of recombinant baculovirus shuttle vector

[0023] The original protein sequence is shown in SEQ ID NO.1, and the GQGSSHHHHHHSSGQG sequence is inserted in the middle of 62-63 amino acids, and the final protein sequence is shown in SEQ ID NO.2. The gene sequence encoding the protein shown in SEQ ID NO.2 is connected to the 4102-4103bp multiple cloning site of the pFast Bacl vector to obtain a recombinant baculovirus shuttle vector.

[0024] The recombinant baculovirus shuttle vector was transformed into Escherichi...

Embodiment 2

[0032] (1) Construction of recombinant baculovirus shuttle vector

[0033] The original protein sequence is shown in SEQ ID NO.1, and the GQGSSHHHHHHSSGQG sequence is inserted in the middle of 112-113 amino acids, and the final protein sequence is shown in SEQ ID NO.3. The gene sequence encoding the protein shown in SEQ ID NO.3 was connected to the pFast Bacl vector to obtain a recombinant baculovirus shuttle vector.

[0034] The recombinant baculovirus shuttle vector was transformed into Escherichia coli DH10Bac competent cells, and transposed with the Bacmid plasmid at a specific point, and screened with kanamycin, gentamicin, tetracycline, IPTG, and X-gal plate blue and white spots, and grew in the dark for 48 hours , Pick the white single colony on the plate and insert it into 5ml LB medium for overnight culture, extract the recombinant Bacmid plasmid, identify the target fragment by PCR, and use the coeruleus colony as a negative control. Such as figure 1 As shown, the ...

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Abstract

The invention discloses a method for efficiently recombining and expressing PCV2, which belongs to the field of biotechnology. The present invention inserts a His affinity tag into the loop region of PCV2, and then uses a bac to bac baculovirus expression system to express and purify the recombinant protein PCV2 with Sf9 cells in a serum-free medium. The method of the present invention obtains the soluble PCV2 protein through the purification of His FF in one step, which avoids the traditional complicated CsCl 2 The purification method of density gradient centrifugation, and the yield is 3 times higher than that mentioned before, which laid the foundation for the development of PCV2 subunit vaccine.

Description

technical field [0001] The invention relates to a method for efficiently recombinantly expressing PCV2, in particular to a method for designing and constructing a eukaryotic expression system for recombinantly expressing PCV2 based on a three-dimensional structure, and belongs to the field of biotechnology. Background technique [0002] PCV2 (porcine circovirus type 2) is the main pathogen of multisystemic wasting syndrome (PMWS) in weaned piglets. Since the outbreak of PMWS in Canadian pig herds in 1991, PMWS has caused great economic losses to the swine industry worldwide. In recent years, PMWS has also been prevalent in pig herds in my country, and the damage caused is very serious. [0003] At present, vaccine immunization is an important means to control PCV2 infection and related diseases. In 2010, my country developed a whole-virus inactivated vaccine, but the ability of PCV2 to proliferate in vitro cells is weak, it is difficult to cultivate, and the final titer of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12N15/866C07K14/01A61K39/12A61P31/20
CPCA61K39/00C07K14/005C12N15/86C12N2710/14043C12N2750/10022C12N2750/10034C12N2750/10051C12N2800/105
Inventor 王峰
Owner BIORTUS BIOSCI