A method for efficiently expressing pcv2cap and pcv3cap fusion proteins
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A fusion protein and protein technology, applied in the field of molecular biology, can solve the problems of complicated purification process and low expression amount of fusion protein, and achieve the effects of high biological activity, high expression efficiency and lower production cost.
Active Publication Date: 2021-11-30
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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[0005] Aiming at the current problems of low expression of PCV2 Cap-PCV3 Cap fusion protein and complicated purification process, the present invention provides a new method for expressing PCV2 Cap and PCV3 Cap fusion protein. Modified, close to the natural virus-encoded protein, with high biological activity
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Embodiment 1
[0040] Example 1 Construction of a rodovirus
[0041] 1. The result of the gene
[0042] Referring to "Establishment of Porcine Ring Virus 2 Sybr Green I Real Time Fluorescent Quantitative PCR Detection Method", "Establishment of Porcine Ring Virus 3 Sybr Green I Real Time Fluorescent Quantitative PCR Detection Method" 2 and 3 The plasmid of the porcine circular virus genome PEASY-BLUNT-PCV2, PEASIY-BLUNT-PCV3, and preserved for -80 ° C.
[0043] Table 1 Purpose Gene PCR primer sequence
[0044] .
[0045] According to Table 1 synthesis primers, PEASY-BLUNT-PCV2 is a template, and the amplification of PCV2 CAP base sequences is performed in HBM-PCV2CAP F, Linker1-PCV2CAP R, and the amplified product is recovered by glue recovery kit. Named 2CAP. The PEASY-BLUNT-PCV3 is a template, and the amplification of PCV3 CAP base sequences in LINKER2-PCV3CAP-PHF, PCV3cap-pHR is primer, and the amplified product is recovered with a glue recovery kit and is named 3 cp. The recombinant sequen...
Embodiment 2
[0080] Example 2 Expression of fusion protein
[0081] P2 recombinant baculoviruses were inoculated into MOI = 1, 0.5, 0.1 viral volume to 2.5 × 10 6 A / MLSF9 cells. 200 μl of the supernatant was charged every 24 hours, and 5 days were collected, and the protein sample was purified, the steps are as follows:
[0082] 1) The collected supernatant 4 ° C, 5000 × g is centrifuged for 30 min to remove cell debris and impurities, and the cultured supernatant is collected;
[0083] 2) Centrifugal cells were centrifuged at 30000 rpm 1 h, harvested precipitation, ie sterile PBS resuscitation;
[0084] 3) Sterile PBS resuspended precipitation to the upper level liquid surface of 10% -30% -50% sucrose density gradient, 35 000 rpm centrifugation 1.5 h for purification, carefully collect white floc between 30% -50% sucrose layer The strip was resuspended with PBS, 30 000 rpm, centrifuged 1.5 h removal of sucrose, collected, 2 ml sterile PBS resuspended, -80 ° C preservation. SDS-PAGE electrop...
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Abstract
The invention provides a method for efficiently expressing PCV2 Cap and PCV3 Cap fusion proteins. First, construct a recombinant baculovirus that efficiently expresses PCV2Cap and PCV3Cap proteins: the nucleic acid sequence of the PCV2Cap protein with the nuclear localization signal cut off and the nucleic acid sequence of the PCV3Cap protein Connection, a hydrophobic flexible protein linker is used in the middle, and a beeline signal peptide sequence is added to the N-terminus of the PCV2Cap and PCV3Cap sequences to promote their secretion and expression. The protein expressed by the recombinant positive baculovirus infected sf9 insect cells undergoes various post-translational modifications, which is close to the natural virus-encoded protein and has high biological activity. Moreover, the baculovirus is highly species-specific and only infects Insect cells are non-infectious to vertebrate cells, and their expression products are safe and reliable, and can be used in follow-up experiments after simple treatment, which is safer and more effective than traditional methods.
Description
Technical field [0001] The present invention belongs to the field of molecular biology, and in particular to a method of efficient expression of PCV2 CAP, PCV3 CAP fusion protein. Background technique [0002] Porcine Circovirus (PCV) mainly infringes wear pigs and fattening pigs, which can cause a variety of pig ring virus-related diseases, and the healthy development of pig industry brings a huge threat. According to antigenicity, pathogenicity, and gene homology, PCV is divided into Porcine Circovirus 1, PCV1, Porcine Circovirus 2 (PORCINE Circovirus 2, PCV2) and Pig Rings Three genotypes of virus 3 (PORCINE CIRCOVIRUS 2, PCV2). It is known that PCV1 has no pathogenicity, while PCV2 has strong pathogenicity, which can cause a variety of porcine circovirus-as solid deseses, PCVADS, which is the most important part of the weakened piggy pig. PostWeaning MultiSystemic Wasting Syndrome, PMWS, the disease is widely existed in our country and the world of pigs, has become a common d...
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