Preparation method of capsid recombinant protein prokaryotic expression of emiliania huxleyi virus

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The invention discloses a preparation method of a capsid recombinant protein prokaryotic expression of emiliania huxleyi virus, comprising the following steps: a) purifying and concentrating the emiliania huxleyi virus; b) designing a primer; c) extracting the DNA; d) performing the PCR amplification on the EhV capsid protein gene of the emiliania huxleyi virus; e) transforming the PCR electrophoresis recovery product to the escherichia coli cells, selecting the cells for clone, extracting the plasmids for enzyme digestion, and then analyzing the PCR product by the electrophoresis and sequencing; f) obtaining the target gene fragment by the double-enzyme digestion, connecting the plasmids, transforming the escherichia coli cells and screening the celss to obtain a recombinant carrier; g) performing small target gene expression; h) performing large target gene expression; and i) detecting the protein expression by SDS-PAGE. The preparation method of the invention can obtain purified recombinant virus capsid protein in large quantity, which can be further used in the preparation of immune antibody, and provide the material foundation for building the immunofluorescence detecting technique for the fast and accurate detection of the host cell cracking rate caused by the infection of the emiliania huxleyi virus.

In order to solve the above problems, the present invention is to provide a method for preparing marine Coccolithophores prokaryotic expression of recombinant virus shell, the method of the present invention can quickly and accurately detect the host cell Coccolithophores virus infection leads to cleavage rate.

To achieve the above object, the present invention adopts the following technical scheme:

A marine Coccolithophores prokaryotic expression of recombinant virus coat preparation method comprises the following steps:

a) marine Coccolithophores virus EhV (isolated from Norwegian waters, presented by the Institute of Biology Department of Microbiology, University of Bergen, Norway) between the host system and the establishment of a stable infection, viral infection 90 hours after taking viral lysate supernatant solution for virus purification and concentration;

b) based on the full-length sequence of marine ball stone design primers EhV algal virus coat protein gene;

c) from step a) of purified virus EhV ocean Coccolithophores extracted DNA;

d) PCR amplification of the ocean Coccolithophores virus coat protein gene EhV;

e) PCR product was recovered electrophoresis connected plasmid transformed into E. coli competent cells, clones were picked, plasmids were extracted using EcoR I and Not I after double digestion electrophoresis and sequencing of PCR products, marine Coccolithophores EhV virus coat protein gene sequence sequencing results see Table 1.

f) to select consistent with the expected size bands were recovered to obtain the target gene fragment by restriction enzyme digestion, connection plasmid, transformed into E. coli competent cells, screened to obtain a recombinant vector;

g) The constructed recombinant expression vector was transformed into E. coli strains, clones were picked up small amounts of target gene expression;

h) picked clones were a lot of target gene expression;

i) the induction of the sample before and after the induction of the sample purified by SDS-PAGE to identify protein expression.

The purification process using SM buffer, whose formula is: 10mmol / L NaCl, 50mmol / L Tris (Tris), IOmmol / L MgSO4 and 0. gelatin (gelatin) (commercially available. in Shanghai Sangon AMERSC0 repackaging), pH 7. 5.

The primer composition using SEQIDN0 1 and SEQIDN0 2; its nucleotide sequence, respectively:.. SEQIDN0]: MCP-F: 5 '-GACGAATTCCTCTGGATTTGGTGGTGG-3' (the underlined EcoRI restriction site);.

The reaction conditions for PCR were: 94 ° C denaturation for 1 minute denaturation 94 ° C after 15 seconds, 55 ° C annealing 30 seconds, 68 ° C extension for 2 minutes 30 amplification cycles, to save at 4 ° C.

The step e) E. coli using DH5ci; said step g) using E. coli BL21 (DE3). (Green Day were purchased from Institute of Biotechnology)

The double digestion using enzymes EcoRI and NotI (both purchased from TaKaRa Biotechnology Co., Ltd. Guangzhou Rui true).

The step e) using plasmid pMD19-T as a carrier (purchased from TaKaRa Biotechnology Co., Ltd. Guangzhou Rui true).

The step f) using pGEX-4T-3 as an expression vector. (Purchased from GE Healthcare Xiamen Egret Long Biotechnology Development Co., Ltd.), a recombinant vector is pGEX-4T-3-EhVMCP, contain claims ocean Coccolithophores recombinant virus coat protein gene DNA sequence 1.

The present invention is a beneficial effect: The invention is based on research and analysis of the major coat protein of the virus (MCP) gene fragment obtained, EhV between double-stranded DNA viruses and other algae varies greatly, therefore, Coccolithophores virus has been divided for algae double-stranded DNA virus family in a new genus (Coccolithovirus). Meanwhile, between different strains of MCP genes conserved regions EhVs some bases variability is higher. Therefore, MCP gene has been used as a new, more stable molecular genetic markers to distinguish between natural communities EhVs different genotypes. Although different genes conserved regions EhVs MCP isolates certain diversity in the nucleic acid sequence, but its similarity to the deduced amino acid sequence corresponding is very high, reaching 99% -100%. Further, such virus has a unique virus different from the infected animals is similar to the way other algae viruses, virus through endocytosis or membrane fusion mode, will include the entire coat protein including a complete viral particle into a host cell . Based EhVs coat protein amino acid homology highly conserved region of the infection and its unique way that MCP can be used as a marker protein detected in natural waters can be EhVs algae and other DNA viruses to distinguish. Thus, by expressing EhVs MCP, established serological methods, will be able to replace the cumbersome TEM method for the presence of natural waters EhVs trace detection, with an estimated prevalence of the host, the cleavage rate and death situation for the rapid and accurate detection Coccolithophores infected host cell lysis rate due to provide a new method. The present invention is set forth in the use of this new technology and recombinant prokaryotic expression technology for some difficult purified virus clone, prepared so that the use of an immunological method of testing the rate of infection and lysis caused by these viruses into host cells possible to assess the settlement via cell death after Coccolithophores total carbon dioxide into the seabed and the release of DMS, an important role in the development of carbon dioxide emissions specifications and comprehensive evaluation Coccolithophores virus in the global climate change provide a theoretical basis for the government.