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Construction method of CHO cell strain by fusing porcine serum protein and porcine circovivus Cap2 protein and application thereof

A cell line and protein technology, applied in the direction of serum albumin, cells modified by introducing foreign genetic material, viruses, etc., can solve the problems of poor pigs, short antibody production time, uneven immune effect, etc.

Active Publication Date: 2017-11-07
TANGSHAN YIAN BIOLOGICAL ENG CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is how to improve porcine circovirus cap2 protein expression in Escherichia coli expression system virus-like particle protein formation inhomogeneous immune effect poor and endotoxin residual high caused pig death defect; or other expression system expression product Defects such as short antibody production time and residual protein causing immune fever

Method used

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  • Construction method of CHO cell strain by fusing porcine serum protein and porcine circovivus Cap2 protein and application thereof
  • Construction method of CHO cell strain by fusing porcine serum protein and porcine circovivus Cap2 protein and application thereof
  • Construction method of CHO cell strain by fusing porcine serum protein and porcine circovivus Cap2 protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1, the acquisition of Pigpsa-PCV2cap2 mutant and its coding gene

[0067] To improve the long-term potency of the isolated wild-type Cap2, the protein sequence of the isolated porcine circular Cap2 was fused to the porcine serum Psa fragment. The specific protein sites of specific fusion proteins are shown in Table 2.

[0068] Table 2 Fusion protein design

[0069] name

[0070] Synthesis of the Pigpsa-PCV2cap2 gene shown in SEQ ID No.1 is to fuse the N-terminus of the Cap2 protein to the porcine Psa fragment. The indicated protein Pigpsa-PCV2cap2.

Embodiment 2

[0071] Embodiment 2, the preparation of Pigpsa-PCV2cap2 expression antigen

[0072] 2.1 Obtaining of recombinant Pigpsa-PCV2cap2 recombinant expression vector

[0073] After the gene was synthesized by the gene synthesis company, it was directly constructed on the PCDNA3.1 vector, and the results were as follows: figure 1 and figure 2 , the successful strain was constructed, inoculated in 100ml ampicillin-containing LB medium and 250ml Erlenmeyer flask, cultured overnight at 37°C on a shaker at 220rpm, and the carrier was extracted with Tiangen non-endotoxic kit. After pvul digestion, use the gel recovery kit to recover the digested products, and the recovered products are ready to be used for transfection of mammalian CHO cells.

[0074] 2.2 Acquisition of fusion expression cell CHO

[0075] The product was purified after pvul digestion, and CHO cells were collected.

[0076] 2.2.1. The day before transfection, inoculate on a 6-well culture plate at a suitable cell densi...

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Abstract

The invention relates to a vaccine production technology in the field of biotechnology, in particular to a CHO cell strain expressing recombinant protein Pigpsa-PCV2cap2 and constructed with a genetic engineering method, and a preparation method and application of the CHO cell strain. The recombinant fusion protein Pigpsa-PCV2cap2 provided by the invention is shown as A1) or A2) as follows: A1) the amino acid sequence is as shown as SEQ ID No. 2; and A2) superseding / missing / adding one or more amino acid residues in the amino acid sequence of the protein of A1) to obtain a protein having Pigpsa-PCV2cap2 activity. The monoclonal cell strain capable of secreting and expressing Pigpsa-PCV2cap2, obtained with the method, has the relatively high expression level of the fusion protein; the fusion protein obtained after His affinity, separation and purification can be bonded with a monoclonal antibody, so as to be better than other current marketing products in the aspects of immunizing animals and generating a neutralizing antibody; the fusion protein can be used for preparing porcine circovirus vaccine, so as to reduce the production cost, and avoid death and immune failure loss caused by endotoxin residues.

Description

technical field [0001] The invention relates to vaccine production technology in the field of biotechnology, in particular to a CHO cell strain expressing recombinant protein Pigpsa-PCV2cap2 constructed by means of genetic engineering, and a preparation method and application thereof. technical background [0002] Porcine circovirus belongs to the genus Circovirus of the family Circoviridae. The size of the virus particle is 14-25nm, with an average diameter of 17nm. , the suspension density in the tissue is 1.37 cm3, and the sedimentation coefficient is 52S, which is the smallest animal virus found so far. The replication of PCV is a rolling circle replication method, which first passes through a double-stranded replication type (RF), and then is transcribed and encoded by the double-stranded replication type. This method of replication is the same as that of chicken infectious anemia virus (CAV). Porcine circovirus (PCVPorcinecircovirus,) is one of the smallest animal vi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/12A61P31/20
CPCA61K39/12C12N15/62C07K14/005C07K14/765C12N2750/10034C12N2750/10022
Inventor 伏显华李润孙爱娟
Owner TANGSHAN YIAN BIOLOGICAL ENG CO LTD
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