Preparation method and application of recombinant EBV gHgL immunogen

An immunogen and protein technology, applied in the field of biomedicine, can solve the problem of low yield and achieve the effect of large expression, good purity and uniformity, and good natural fidelity

Active Publication Date: 2020-05-15
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently established gHgL express...

Method used

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  • Preparation method and application of recombinant EBV gHgL immunogen
  • Preparation method and application of recombinant EBV gHgL immunogen
  • Preparation method and application of recombinant EBV gHgL immunogen

Examples

Experimental program
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Embodiment 1

[0027] The construction of embodiment 1 recombinant gH and gL expression vector

[0028] Using the gH and gL cDNAs synthesized by Jinweizhi Biotechnology Co., Ltd. with codon-optimized codons according to the codon preference of Drosophila as templates, the extracellular domain of gH and the full length of gL were obtained by PCR technology (see figure 1 ) DNA fragment.

[0029] The primers used in the vector construction process are:

[0030] pMT EBV gH Bgl II Forward: GAAGATCTGCCTCCCTCTCCGAGGTCAAAC, SEQ ID NO. 1;

[0031] pMT EBV gH Xba I Reverse: GCTCTAGAGTGGGCACGTTCTTCGTAGAG, SEQ ID NO. 2;

[0032] pMT EBV gL Bgl II Forward: GAAGATCTAACTGGGCCTATCCGTGCTGTCAC, SEQ ID NO. 3;

[0033] pMT EBV gL Xba I Reverse: GCTCTAGAGCCACCTCGATGCCAAGCGTAC, SEQ ID NO.4.

[0034] The PCR reaction system is as follows:

[0035]

[0036] The reaction procedure is as follows:

[0037]

[0038] The two target gene fragments were respectively connected to the modified pMT / Bip / TEV-HisA v...

Embodiment 2

[0045] Example 2 Three-plasmid co-transfection of Drosophila melanogaster S2 cells and screening of S2 stable cell lines expressing target protein

[0046] S2 cells were plated in T25 culture flask one day in advance, and co-transfection was carried out by liposome method when the confluency of the cells reached 60-70%. The transfection step was carried out according to the instructions of the transfection reagent Cellfectin II, the mass ratio of the two recombinant plasmids and the resistance selection plasmid pCoBlast was 19:19:1, and the ratio of gH and gL could be changed to obtain optimal expression, but the two Together, the ratio to the screening plasmid was 38:1. Place the transfected cells in a 27°C biochemical incubator and incubate for 5 hours, discard the transfection solution, add 5 mL of fresh SFX-Insect medium, continue to cultivate for 48 hours, discard the original medium, and add The SFX-Insect medium with blasticidin (final concentration: 25 μg / mL) was used...

Embodiment 3

[0047] Expression and purification of embodiment 3 recombinant gHgL

[0048] The selected T25 cultured S2 stable cell line was expanded into a T75 culture flask with a volume ratio of 1:5, and further expanded into a 500mL spinner bottle with a volume ratio of 1:5 for culture, at 27°C, 120- 130rpm, culture for 2-3 days, when the cell density reaches 4-6×10 6 , adding copper sulfate at a final concentration of 0.5 mM to induce the expression of the recombinant protein, and collecting the cell supernatant after 3 days. Pour the S2 cells into a 500mL centrifuge bucket, centrifuge at 4000rpm, 4°C for 15min. The cell supernatant was collected, filtered through a 0.22 μm microporous membrane, transferred to an Amicon Stirred Cell 8003 ultrafiltration cup for concentration, compressed to about 50 mL, and combined with a nickel column buffer (50 mM Tris-HCl, 500 mM NaCl, pH8.0) was diluted 10 times, continued to compress to 40mL, collected the protein solution into a high-speed cent...

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Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a recombinant EBV gHgL immunogen. The polymerase chain reaction PCR technology is used to obtain gene fragments of gH and gL, the two target fragments are separately cloned into an insect expression vector pMT, endotoxin is removed through plasmid large-scale extraction, then the two target fragments and a resistance screening plasmid pCoBlast are co-transfected into Drosophila melanogaster Schneider2 cells, blasticidin is used for positive clone screening, and stable S2 cell lines for efficiently secreting and expressing gHgL are screened out. After expanded culture, the gHgL with high uniformity, high yield and high natural fidelity is obtained through two steps of purification of nickel column affinity chromatography and gel filtration chromatography. The interaction between the gHgL and a receptor EphA2 is verified by the micro-scale thermophoresis technology, and thereby the gHgL is suitable as an immunogen for the research and development of EBV subunit vaccines, and combined vaccines with gB and gp350.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of recombinant EBV gHgL immunogen. Background technique [0002] Epstein-Barr virus (EBV), referred to as EB virus, belongs to the Gammaherpesvirus subfamily, can infect about 95% of the world's population, and is the earliest virus associated with tumors. It is associated with a variety of human diseases, especially malignant tumors, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric cancer. It is estimated that there are about 200,000 new cases of EBV-related cancer patients in the world every year, and about 140,000 patients die from it. Therefore, safe and effective vaccines are urgently needed to block EBV infection. In view of the characteristics of lifelong EBV infection and its high carcinogenicity, there may be safety problems in attenuated live vaccines, and vaccine research and ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C07K14/03A61K39/245A61P31/22
CPCC12N15/85C07K14/005C07K16/085A61K39/12A61P31/22C12N2800/105C12N2710/16222C07K2317/76C12N2710/16234
Inventor 段良伟王辉郑前前牛玉娜牛志国张佳璐刘梦楠
Owner XINXIANG MEDICAL UNIV
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