Nucleic acid construct and method for preparing coxsackievirus A16 virus-like particles

A Coxsackie virus and nucleic acid construct technology, applied in the fields of virology, immunology, and molecular biology, can solve the problems of protein expression and misfolding

Active Publication Date: 2018-10-09
SA BIOTECH (SUZHOU) PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The inventors have found in previous studies that if the four proteins / polypeptides (VP1, VP2, VP3 and VP4) constituting the HFMD virus Coxsackie virus A16 capsid protein are expressed separately, the problem is: or this Several proteins could not be expressed separately in E. coli, or after being expressed separately, they were in a state of misfolding (as demonstrated by the experimental results of Comparative Example 1 below)

Method used

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  • Nucleic acid construct and method for preparing coxsackievirus A16 virus-like particles
  • Nucleic acid construct and method for preparing coxsackievirus A16 virus-like particles
  • Nucleic acid construct and method for preparing coxsackievirus A16 virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1: VP1, VP2, VP3, VP4 tandem soluble co-expression recombinant vector construction

[0124] The full length of the Coxsackie virus type A16 capsid protein (VP1, VP2, VP3 and VP4) gene, which was codon optimized by E. coli, was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (SEQ ID NO: 5-8). And synthesize the primers shown in Table 1 above.

[0125] The full-length fragments of the Coxsackie virus A16 capsid protein (VP1, VP2, VP3, VP4) gene synthesized previously were used as the PCR reaction template. The primer sequences and names used are shown in Table 1 above.

[0126] Coxsackie virus A16-VP1F was used as the forward primer, Coxsackie virus A16-VP1R was used as the reverse primer, and VP1 was used as a PCR template to amplify the Coxsackie virus A16 capsid protein VP1 gene; Saatchi virus A16-VP2F was used as forward primer, Coxsackie virus A16-VP2R was used as reverse primer, and VP2 was used as PCR template to amplify the Coxsackie virus A16 capsid p...

Embodiment 2

[0140] Example 2: Tandem co-expression of Coxsackie virus A16 capsid protein (VP1, VP2, VP3, VP4)

[0141] Transform the pET-Q-CoxA16-VP3124 plasmid prepared in Example 1 into 40 μl of competent E. coli BL21 (DE3) prepared by the calcium chloride method, and spread it on kanamycin-resistant solid LB medium, Incubate at 37°C for 10-12 hours until a single colony is clearly identifiable. Pick a single colony into a test tube containing 4ml of kanamycin-resistant liquid LB medium, culture it with shaking at 230 rpm at 37°C for 12 hours, and take 1 mL of bacterial solution from it and freeze-dry it at -80°C for storage.

[0142] Take out the Escherichia coli strain with recombinant plasmid pET-Q-CoxA16-VP3124 from -80℃, insert 50ml LB liquid medium resistant to kanamycin, 230rpm, 37℃, cultivate for about 12 hours, then transfer Connect to 1L LB liquid medium, express a lot at 37℃, wait for OD 600 After the value reached 0.6, 0.1 mM IPTG was added to induce protein expression, overnig...

Embodiment 3

[0155] Example 3: Affinity color of Coxsackie virus A16 capsid protein (VP1, VP2, VP3, VP4) with SUMO tag Spectrum purification preparation

[0156] The bacterial cells prepared in Example 2 were resuspended in a ratio of 1 g bacterial cells to 10 mL lysate (20mM Tris buffer pH 7.2, 300mM NaCl), and the bacterial cells were disrupted 4 times with a homogenizer at a pressure of 700 bar. JA-14 rotor 13500rpm (28000g), centrifugation for 40 minutes, the supernatant was collected, and the supernatant was detected by 15% SDS-polyacrylamide gel electrophoresis. At this time, the SUMO-labeled Coxsackie virus A16 capsid protein ( The purity of SUMO-VP1, SUMO-VP2, SUMO-VP3, SUMO-VP4) is about 10%.

[0157] After the supernatant was filtered with a 0.45 μm pore filter membrane, the XK 26 / 60 column (GE Healthcare Life Sciences) packed with His beads (GE Healthcare Life Sciences) was used for chromatographic purification.

[0158] Instrument system: AKTA purified preparative liquid chromatogr...

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Abstract

The invention belongs to the fields of molecular biology, virology and immunology, and relates to a nucleic acid construct and a method for preparing coxsackievirus A16 virus-like particles. The invention also relates to recombinant vectors and recombinant host cells comprising the nucleic acid construct. The invention further relates to a method and a purification method for co-expressing coxsackievirus A16 type capsid proteins VP1, VP2, VP3 and VP4 in series, a method for preparing the coxsackievirus A16 type virus-like particles, and the like. Further, the invention relates to the coxsackievirus A16 type virus-like particles and a pharmaceutical composition containing the same, such as a vaccine. By adopting the nucleic acid construct and the method, soluble co-expression of the capsidproteins of a human hand-foot-and-mouth disease virus (coxsackievirus A16 type) in escherichia coli is realized, and the expression quantity is relatively high; finally obtained interest proteins account for about 10% of the total soluble proteins in bacteria; and the coxsackievirus A16 virus-like particles comprising the interest proteins are highly similar to native coxsackievirus A16 conformation.

Description

Technical field [0001] The invention belongs to the fields of molecular biology, virology and immunology, and relates to a nucleic acid construct and method for preparing Coxsackie virus A16 type virus-like particles. The present invention also relates to a recombinant vector and a recombinant host cell containing the nucleic acid construct. The present invention also relates to a method for co-expressing Coxsackie virus A16 capsid proteins VP1, VP2, VP3 and VP4 in series and a purification method, a method for preparing Coxsackie virus A16 virus-like particles, and a method Method for preparing hand, foot and mouth disease vaccine. In addition, the present invention also relates to the preparation of the above-mentioned nucleic acid construct, recombinant vector or recombinant host cell for the treatment and / or prevention and / or adjuvant treatment of Coxsackie virus A16 infection or diseases caused by Coxsackie virus A16 infection (such as hand and foot Oral disease) medicine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N7/04A61K39/125A61P31/14
CPCA61K39/12A61K2039/523A61K2039/5258C07K14/005C07K2319/00C12N7/00C12N15/70C12N2770/32023C12N2770/32034C12N2770/32051
Inventor 袁于人
Owner SA BIOTECH (SUZHOU) PTE LTD
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