Pseudovirion containing hepatitis c virus RNA (Ribonucleic Acid) fragment and preparation method thereof

Abstract

The invention provides a pseudovirion containing hepatitis c virus (HCV) RNA (Ribonucleic Acid) fragment and preparation method thereof which are applied in the field of biomedical clinical vitro diagnostic. The pseudovirion is a RNA-protein complexes that MS2 bacteriophage capsid protein wraps the hepatitis c virus, and the complexes is icosahedral; the method comprises the following steps of designing and synthesizing primer to obtain target gene MS2 by overlapping and splicing PCR (Polymerase Chain Reaction) method; connecting the target gene MS2 to plasmid pET-32a (+) to obtain recombinant plasmid pET-MS2; connecting the recombinant plasmid pET-MS2 and the HCV fragment to obtain pET-MS2-HCV recombinant plasmid; the obtained pET-MS2-HCV recombinant plasmid is introduced into escherichia coli for prokaryotic expression; releasing virus-like particles by adopting ultrasonication and the obtained virus-like particles are the pseudovirion containing the hepatitis c virus RNA fragment. The pseudovirion preparation method provided by the invention has the advantages that the preparation method is simple, the operation is easy, the obtained pseudovirus is high in purity, and the pseudovirion can be used as standard and quality control material of detection of RT-PCR (Reverse Transcription-Polymerase Chain Reaction), is good in stability, has no infectivity, has RNase-resistant, and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a pseudovirus particle containing hepatitis C virus RNA fragments and a preparation method thereof. Background technique [0002] Hepatitis C is a blood-borne disease mainly caused by hepatitis C virus (HCV) infection. Some patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC). There is no effective cure, so early and accurate diagnosis of HCV infection is of great significance. [0003] HCV is a group of viruses that cause hepatitis in humans, and its genome is highly heterogeneous. According to the degree of difference in the nucleotide sequence of the HCV genome, HCV can be divided into six main genotypes and different subtypes. According to the internationally accepted method, the HCV genotype is expressed in Arabic numerals. The gene subtypes are represented by lowercase English letters (such as 1a, 2b, 3c, etc.). HCV types 1 to 3 are widel...

AI Technical Summary

Problems solved by technology

HCV RNA transcribed in vivo or in vitro can be used as a positive control for reverse transcription; however, because it does not have a viral shell, it is not suitable as a positive control in the step of extracting viral RNA, and it is easily degraded by RNase, which is ubiquitous in the environment, and is not easy to save

The advantage of using a plasmid as a positive control is that it is easy to prepare, but because the plasmid is DNA, not RNA, it can only be used as a positive control for the PCR amplification step, but cannot be used as a control for RNA extraction and reverse transcription

As for the chemical synthesis method, the cost is very high, and it is easy to degrade without the protection of the nucleocapsid, so it is not suitable for large-scale production

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