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Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof

A technology of human cytomegalovirus and fusion protein, which is applied in the field of human cytomegalovirus immunogen fusion protein and its preparation, can solve the problems of poor effectiveness of human cytomegalovirus vaccine, achieve good clinical application prospects, good immune effect, immune strong effect

Active Publication Date: 2012-12-12
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of poor effectiveness of existing human cytomegalovirus vaccines, the present invention provides a human cytomegalovirus immunogen fusion protein

Method used

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  • Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof
  • Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof
  • Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Construction of embodiment 1pET42b / FljB expression vector

[0071] Take the pET42b plasmid and the nucleotides encoding flagellin FljB, and clone the nucleotides encoding flagellin FljB into the pET42b plasmid with NdeI and BamHI endonucleases to obtain the pET42b / FljB vector.

[0072] Ligation transformation: The recovered target fragment was ligated with the pMD19-T simple vector at a molar ratio of 3:1 at 16°C for 1 hour, and then transformed into Escherichia coli TOP10 competent cells (CaCl 2 prepared by method). The specific method for transforming E. coli competent cells with the linker: thaw the TOP10 competent cells on ice; add the ligation reaction (4 μl), rotate gently, and then incubate on ice for 5 minutes; place the test tube in a water bath at 42 Heat shock at °C for 30 seconds; cool the tube on ice for at least 2 minutes; add SOC medium (400 μl) to the cells and shake at 37°C for 1 hour.

[0073] Screening: Use two LB agar kanamycin (50 μg / ml) culture d...

Embodiment 2

[0075] Example 2FljB-AD1 fusion protein expression vector construction and its expression and purification

[0076] 1. Construction of Fusion Protein Expression Vector

[0077] The pET42b / FljB prepared in Example 1 and the synthesized AD1 nucleotide sequence were subjected to SpeI / KpnI double enzyme digestion respectively. The digested product was subjected to agarose electrophoresis, and the 260bp AD1 and about 7.2kb pET42b / FljB fragments were excised, purified with Qiagen gel recovery kit, and ligated with the pMD19-T simple vector at a molar ratio of 3:1 at 16°C for 1 hour. The ligation product was transformed into Escherichia coli TOP10 competent cells. Positive clones were screened on a kanamycin-resistant LB plate (same as in Example 1), and pET42b / FljB-AD1 was obtained by plasmid extraction and restriction restriction plasmid identification, and the results were as follows figure 2 , indicating that the pET426b / FljB-AD1 vector was successfully constructed, and its st...

Embodiment 3

[0083] Example 3 Construction of FljB-AD2 fusion protein expression vector and its expression and purification

[0084] 1. Construction of Fusion Protein Expression Vector

[0085] The pET42b / FljB and pUC57 (containing the synthetic AD2 sequence SEQ ID NO: 3) constructed in Example 1 were subjected to SpeI / KpnI double enzyme digestion respectively. The digested product was subjected to agarose electrophoresis, and the 318bp AD2 and about 7.2kb pET42b / FljB fragments were excised and purified with a Qiagen gel extraction kit. The ligation reaction was the same as in Example 1. The ligation product was transformed into Escherichia coli TOP10 competent cells. Positive clones were screened on the kanamycin-resistant LB plate (same as in Example 1), and pET42b / FljB-AD2 was obtained by plasmid extraction and restriction restriction plasmid identification, and the results were as follows Image 6 , indicating that the pET42b / FljB-AD2 vector was successfully constructed, and its stru...

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Abstract

The invention discloses a human cytomegalo virus immunogen fusion protein which comprises flagellin, human cytomegalo virus envelope glycoprotein or polypeptide segment; the invention discloses deoxyribonucleic acid (DNA) molecules used for encoding the fusion protein, recombinant plasmid containing the DNA molecules, and a recombinant expression vector; the invention discloses a preparation method and usage of the fusion protein; and the invention also discloses a human cytomegalo virus vaccine. The fusion protein is high in immunogenicity and can be used for stimulating an organism to generate a great deal of cytomegalo virus antibodies and preparing the vaccine for preventing diseases caused by cytomegalo virus infection and is good in clinical application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a human cytomegalovirus immunogen fusion protein and a preparation method thereof. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the double-stranded DNA virus of the β-herpesvirinae subfamily, and is the most common opportunistic infection virus, and its infection is species-specific. The infection rate of HCMV showed obvious differences in different places and ages. In developed countries, the positive rate of HCMV antibodies is 50% to 80%. In developing countries, 80% of children are infected with HCMV before the age of 3, and the HCMV infection rate can even reach 100% in adults. The infection rate among Chinese population is as high as 86%-96%. Most healthy people have subclinical infection or latent infection without obvious clinical symptoms, and some patients may have symptoms similar to infectious mononucleosis. However, HCMV infection is inde...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/245A61P31/22
CPCA61K39/12A61K2039/55516A61P31/22C07K14/005C07K14/255C07K2319/00C12N2710/16122C12N2710/16134
Inventor 刘兰军武志强李小娇葛永红
Owner CHENGDU RONGSHENG PHARMA
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