Method for preparing MERS RNA nucleic acid reference
A reference product and nucleic acid technology, applied in the field of genetic engineering, can solve problems such as false negatives, unstable properties, inaccurate detection reactions, etc., and achieve the effect of cost saving and high stability
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Embodiment 1
[0056] Example 1: Construction of lentiviral expression vector pEB-copGFP(T2A)PURO-upE-1a or pEB-copGFP(T2A)PURO-1bN
[0057] In this example, MERS upE and ORF1a RNA and ORF1b and N gene RNA packaged with lentiviral particles were prepared by the following method:
[0058] S1. Obtain the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and clone the two sequences.
[0059] S2. Select genes upE and ORF1a commonly used in nucleic acid detection of MERS, as well as ORF1b and N genes, entrust a gene synthesis company to synthesize, and add restriction endonuclease sites of XbaI and BamHI at both ends.
[0060] S3. The lentiviral packaging vectors of four plasmids (including lentiviral expression vector: pEB-copGFP(T2A) PURO and three packaging plasmids: VSV-G, PLP1, PLP2) were purchased from a gene company. The synthetic MERS upE and ORF1a genes and ORF1b and N genes were connected to the lentiviral expression vector pEB-copGFP(T2A)PURO. The specific enzyme-cut ligatio...
Embodiment 2
[0073] Embodiment 2: the expression of two kinds of pseudovirus particles that packaged MERS upE and ORF1a RNA and ORF1b and N gene RNA
[0074] The lentiviral packaging system used in the present invention is the third-generation four-plasmid packaging system, which has an expression vector and three packaging plasmids: the packaging system puts the Rev gene on a single plasmid in order to prevent homologous recombination to produce self-replicating virus; the second plasmid expresses Gag and Pol structural proteins; the third plasmid uses the herpetic stomatitis virus glycoprotein VSV-G to replace its own envelope protein Env, compared with the first generation two-plasmid packaging system and the second The three-generation plasmid packaging system has higher safety. The construction process of pseudoviral particles is as follows: construct the foreign gene into the lentiviral expression vector, and transfect the cells with three packaging plasmids VSV-G, PLP1, and PLP2. Th...
Embodiment 3
[0082] Example 3: Verification of pseudovirus particles packaged with MERS upE and ORF1a RNA
[0083] Firstly, the samples of pseudovirus particles were prepared and then observed by transmission electron microscope (TEM). The specific process is as follows:
[0084] P1. Place the copper mesh in 20 μL microdroplets of pseudovirus particles packaged with MERS upE and ORF1a RNA. After 10 minutes of adsorption, take out the copper mesh and negatively decontaminate it with 20 μL of 2% phosphotungstic acid (PTA pH 6.8). After dyeing and fixing the sample for 30s, the copper grid was taken out, placed on a filter paper sheet to dry overnight, and then observed with a transmission electron microscope. The result is as figure 1 shown.
[0085] P2, then verify the pseudoviral particles packaged with MERS upE and ORF1a RNA with fluorescent quantitative PCR.
[0086] P3. Take 30 μL of concentrated pseudoviral particles and add 110 μL of sterile water to make up to 140 μL, and perform...
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