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Preparation method of pseudovirus

A pseudovirus and carrier technology, applied in the field of pseudovirus preparation, can solve the problems of low purity, complex pseudovirus purification process, and low packaging efficiency of pseudoviruses, and achieve improved purification efficiency, enhanced long-term storage stability, and packaging efficiency Improved effect

Active Publication Date: 2021-08-17
INTEGRATED BIOSYSTEMS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the prior art, the method of using armored RNA to express and package the pseudovirus packaging efficiency is not high, and the pseudovirus purification process is complicated and the purity is not high.

Method used

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  • Preparation method of pseudovirus
  • Preparation method of pseudovirus
  • Preparation method of pseudovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A kind of preparation method of embodiment 1 pseudovirus

[0061] (1) Construction of pZSR-IPC vector

[0062] The insert sequence SEQ ID NO.12 (including the mature protein SEQ ID NO.7) was designed at the Nco I / HindIII site on the pET28a vector. The specific vector sequence after the insert sequence is shown in SEQ ID NO.16. This part was entrusted to Wuhan Jinkairui Bioengineering Co., Ltd. Ltd Synthetics. SEQ ID NO.13 (including DNA sequence SEQ ID NO.2 expressing capsid protein, His tag SEQ ID NO.9, packaging recognition site SEQ ID NO.8 and target sequence SEQ ID NO.10) was cloned into the synthetic pET28a vector HindIII / XhoI site of SEQ ID NO.16. Prepare enzyme digestion system (50 μL) according to the following table:

[0063]

[0064] After digestion at 37°C overnight, the gel was recovered and purified.

[0065] Prepare the purified vector and fragments according to the following system:

[0066] Reagent Volume (μL) T4 buffer 2 C...

Embodiment 2

[0097] The long-term storage stability detection of embodiment 2 pseudoviruses

[0098] Select the pseudovirus that embodiment 1 obtains to carry out following experiment:

[0099] Place them at 37°C for 7d, 14d, 30d, and 60d respectively, then perform sample processing, one-step reverse transcription fluorescence quantitative PCR detection, and store the control pseudovirus at -20°C, and compare the changes in the Ct values ​​of the two.

[0100] The stability test results are shown in Figure 4 : The change trend of Ct value after storage at 37°C for different time shows that the prepared virus-like particles can be stored at 37°C for at least 60 days with good stability.

Embodiment 3

[0101] The homogeneity test of embodiment 3 pseudoviruses

[0102] Divide the pseudoviruses into 8 tubes, place them at 4°C for 7 days, and do one-step reverse transcription fluorescent quantitative PCR at the same time, see the results in Figure 5 , the obtained CV value is shown in the following table, indicating that the pseudovirus has better uniformity;

[0103]

[0104]

[0105] It can be seen from the above table that the CV value of the pseudovirus is 4.068%, and the uniformity is good.

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Abstract

The invention provides a preparation method of a pseudovirus, an existing vector is modified, the modified vector comprises optimized sequences of capsid protein genes of escherichia coli MS2 bacteriophage, wherein the sequences are one or more of SEQ ID NO.1-6, and the modified vector is used for transforming bacteria and expressing to obtain the pseudovirus with higher packaging efficiency. The long-term storage stability of the pseudovirus is enhanced, and the pseudovirus can be freeze-dried and can be stored at room temperature for a longer time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a pseudovirus. Background technique [0002] Armored RNA (Armored RNA) technology is a new preparation technology for RNA quality control products. This technology solves the problem of poor stability of traditional RNA quality control products, the hidden danger of biological infection, and the purpose of monitoring nucleic acid extraction and reverse transcription process. And other issues. The principle of armored RNA technology is to package specific RNA sequences in protective protein or polymeric molecular shells through RNA viruses or other biotechnology, which can stabilize and protect nucleic acids from nuclease degradation. Common implementation methods include cloning the sequence of the capsid protein gene of Escherichia coli MS2 phage and the foreign gene into an expression vector, which can transcribe the foreign gene into RNA, and us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/34C12N7/00
CPCC12N15/70C12N7/00C07K14/005C12N2795/00021C12N2795/00022Y02A50/30
Inventor 金鑫浩张泽云张明程高静张瑜贾欣月蔡亦梅任鲁风
Owner INTEGRATED BIOSYSTEMS CO LTD
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