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Prawn virus expression system for delivering and expressing exogenous genes in prawns

An expression system and a technology for exogenous genes, applied in the field of genetic engineering, can solve the problems of low tropism, achieve high-efficiency expression and improve biological safety.

Pending Publication Date: 2022-04-08
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the strict host specificity of viruses, the above-mentioned mammalian virus-mediated gene transfer and expression systems have extremely low tropism in shrimp tissues and cells

Method used

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  • Prawn virus expression system for delivering and expressing exogenous genes in prawns
  • Prawn virus expression system for delivering and expressing exogenous genes in prawns
  • Prawn virus expression system for delivering and expressing exogenous genes in prawns

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Cloning of full-length genomic DNA of prawn IHHNV virus and construction of circularized plasmid pUC19-IHHNV.

[0047] Design specific primers, two fragments ( figure 1 ), the full-length genomic DNA of the prawn IHHNV virus can be spliced ​​after sequencing, which is 3833bp. Then, large-fragment homologous recombination (Gibson Ultra Kit), the above two cloned fragments were connected with the linearized pUC19 plasmid, and the pUC19 circularized plasmid of the prawn IHHNV genomic DNA was constructed: pUC19-IHHNV ( Figure 2-Figure 4 ), thereby realizing the amplification and purification of the prawn virus IHHNV genomic DNA in bacteria.

Embodiment 2

[0048] Example 2: Construction of the shrimp virus expression plasmid pUC19-IHHNV-PH-GUS based on the whole genome of the shrimp IHHNV virus.

[0049] pFastBac TM 1-GUS is the template ( Figure 5 ), the PH-GUS-MCS-SV40 pA fragment (2383bp) was amplified by PCR. The left and right ends of the PCR-amplified PH-GUS-MCS fragment have homology arms (2413bp) of 15bp respectively ( Figure 6 ). Then, using the seamless cloning kit of abm company, the PH-GUS-MCS fragment was inserted into the pUC19-IHHNV plasmid linearized by double-enzyme digestion, and a shrimp virus expression plasmid with PH as the promoter and GUS as the reporter gene was successfully constructed: pUC19-IHHNV-PH-GUS ( Figure 7-Figure 8 ).

Embodiment 3

[0050] Example 3: Shrimp IHHNV virus particles (pUC19-IHHNV-PH-GUS) were successfully packaged using Sf9 cells.

[0051] The viral expression plasmid pUC19-IHHNV-PH-GUS was transfected into insect Sf9 cells alone, and it was found that the expression of the GUS gene could not be detected ( Figure 9 ), but the medium supernatant of Sf9 cells after transfection can successfully infect Sf9 cells, and icosahedral virus particles similar to prawn IHHNV were observed in Sf9 cells after infection and in the medium supernatant. about 50-80nm ( Figure 10-Figure 11 ).

[0052] The above results show that the recombinant expression plasmid pUC19-IHHNV-PH-GUS based on shrimp IHHNV virus can be transfected into Sf9 cells alone, and the virus particles of pUC19-IHHNV-PH-GUS can be packaged. Regarding the problem that the expression of the GUS gene could not be detected in sf9 cells after transfection and infection, we believe that the PH promoter is a late promoter of insect baculovirus...

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Abstract

The invention provides a prawn virus expression system for delivering and expressing an exogenous gene in a prawn cell, which comprises a recombinant plasmid for expressing the exogenous gene in the prawn cell, the recombinant plasmid comprises genome DNA (deoxyribonucleic acid) of prawn IHHNV virus, a promoter PH (potential of hydrogen) of insect polyhedrosis protein gene, a polyclone restriction enzyme cutting site MCS (multi-cloning restriction enzyme cutting site) for inserting an exogenous gene and an SV40 polyadenylic acid signal pA. The expression system further comprises an insect baculovirus recombinant expression plasmid and an insect Sf9 packaging cell, wherein the gene of the WSSV envelope protein VP28 is inserted into the insect baculovirus recombinant expression plasmid. The prawn virus expression system provided by the invention can efficiently express exogenous genes in prawn cells, and all plasmid DNA genetic information in the expression system is free out of chromosomes of the prawn cells and cannot be integrated into genomes of the prawn cells, so that the biological safety of the system is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a prawn virus expression system for delivering and expressing exogenous genes in prawn cells. Background technique [0002] Shrimp is an important economic species for marine aquaculture. However, with the continuous expansion of the aquaculture scale, the water quality of the aquaculture environment continues to deteriorate, resulting in more and more serious disease problems in prawn aquaculture. Especially frequent outbreaks of viral diseases have caused huge losses to the shrimp farming industry due to the lack of effective control measures. In order to meet the needs of the isolation and purification of shrimp viruses, the development of diagnostic reagents and nucleic acid vaccines, and the research on the pathogenic mechanism of shrimp virus diseases, it is urgent to establish immortal cell lines of shrimp. [0003] However, due to the lack of effe...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/56
Inventor 郭华荣陶亦文
Owner OCEAN UNIV OF CHINA
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