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Novel genetically engineered subunit vaccine for newcastle disease virus

A newcastle disease virus and amino acid technology, applied in genetic engineering, vaccines, viruses, etc., can solve the problems of difficult cultivation and low virus titer, and achieve the effects of easy purification, reduced complexity, and high safety

Inactive Publication Date: 2019-05-10
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for efficient extraction of specific desired genetic material (DNA) from cultured cells without damaging their DNA or causing them to lose its function during cultivating processes. It achieves these technical benefits through enhancing the yield and efficiency of producing biological products such as therapeutics that are meant to treat diseases caused by inherited defective genes.

Problems solved by technology

This patented technical problem addressed in this patents relates to preventing new castle diseases from spread through commercial avian fluids such as water or air condition systems used for transportation purposes during winter months when birds move around freely within their host range. Current methods involve killing them before they reach market because these treatments may also kill other animals nearby but there could still be harmful consequences if administered too much at once.

Method used

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  • Novel genetically engineered subunit vaccine for newcastle disease virus
  • Novel genetically engineered subunit vaccine for newcastle disease virus
  • Novel genetically engineered subunit vaccine for newcastle disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-HN-GS

[0114] 1. The codon-optimized HN gene was obtained from Nanjing GenScript Biotechnology Co., Ltd., and cloned into the pUC-57 vector to construct the pUC-HN plasmid vector. The optimized HN gene sequence is shown in SEQ ID NO:1.

[0115] 2. HN gene amplification uses pUC-HN as a template, HN-F, HN-R as primers to carry out PCR amplification (gene sequences of HN-F, HN-R are shown in SEQ ID NO.5,6), amplified See Table 1 for the augmentation system. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0116] Table 1 HN gene amplification system

[0117]

[0118] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a band appeared at th...

Embodiment 2

[0129] Example 2 Construction of recombinant eukaryotic expression vector pCI-F-GS.

[0130] 1. GenScript synthesized the codon-optimized F gene and cloned it into the pUC-57 vector to construct the pUC-F plasmid vector. The optimized F gene sequence is shown in SEQ ID NO:3.

[0131] 2. F gene amplification Use pUC-F as a template, and F-F and F-R as primers for PCR amplification (the gene sequences of F-F and F-R are shown in SEQ ID NO.7 and 8). The amplification system is shown in Table 5. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0132] Table 5 F gene amplification system

[0133]

[0134] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as image 3 As shown, a band appeared at the position of 1.5kbp, indicating that the targe...

Embodiment 3

[0146] Example 3: Construction and screening of recombinant CHO cells expressing HN protein

[0147] 1. Cell Transfection

[0148] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0149] 1.2 Plasmid and cell mixing Take 5ug of the pCI-HN-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0150] 1.3 Electric transfer to 280V 20ms for 2 pulses. After the electric shock is completed, immediately transfer the cells to a shaker flask for suspension cul...

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Abstract

The application provides an immune composition and a genetically engineered subunit protein vaccine. The vaccine includes newcastle disease virus HN protein encoded by the nucleic acid molecule shownin SEQIDNO:1 or nucleic acid molecule with the nucleic acid sequence has more than 95% similarity with that of the nucleic acid molecule shown in SEQIDNO:1, and newcastle disease virus F protein encoded by the nucleic acid molecule shown in SEQIDNO:3 or nucleic acid molecule with the nucleic acid sequence has more than 95% similarity with that of the nucleic acid molecule shown in SEQIDNO:3. The vaccine adopts CHO cell eukaryotic expression, protein glycosylation is thorough, the antigen protein immunogenicity is good, the expression quantity is very high and up to (2-3) g/L, recombinant cellscan be subjected to large-scale suspension culture, the complexity of vaccine preparation is reduced greatly and the production cost is reduced.

Description

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Claims

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Application Information

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Owner 苏州世诺生物技术有限公司
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