阅读说明：本技术 一种内含汉城病毒g2蛋白基因装甲rna标准物质及制备方法 (Armored RNA standard substance containing Hancheng virus G2 protein gene and preparation method thereof ) 是由 马思杰 胡群 童淑梅 邹春颖 谢东华 于 2020-04-26 设计创作，主要内容包括：本发明公开了一种内含汉城病毒G2蛋白基因装甲RNA标准物质及制备方法,该RNA标准物质利用噬菌体MS2衣壳蛋白能够抵抗外界环境中核糖核酸酶的降解,对其内部RNA片段起保护作的特性。采用原核表达技术表达MS2噬菌体衣壳蛋白包装汉城病毒部分片段基因,制备成稳定、基于汉城病毒G2囊膜蛋白部分片段序列为靶标的装甲RNA标准物质,适用于汉城病毒核酸检测方法的质量控制。本发明制备的汉城病毒装甲RNA标准物质,具备稳定、无生物安全危害、耐RNase酶降解等特点,便于储存、运输和使用,可对汉城病毒检测的病毒提取、RT-PCR全过程进行质量控制,操作简单、安全有效。(The invention discloses an armored RNA standard substance containing Hancheng virus G2 protein genes and a preparation method thereof, wherein the RNA standard substance utilizes the characteristic that phage MS2 capsid protein can resist the degradation of ribonuclease in the external environment and protect the RNA fragments in the RNA standard substance. The MS2 phage capsid protein is expressed by adopting a prokaryotic expression technology to package partial fragment genes of the Hancheng virus, and the armored RNA standard substance which is stable and takes the partial fragment sequence of the envelope protein of the Hancheng virus G2 as a target is prepared, and is suitable for the quality control of the Hancheng virus nucleic acid detection method. The Hancheng virus armored RNA standard substance prepared by the invention has the characteristics of stability, no biological safety hazard, RNase degradation resistance and the like, is convenient to store, transport and use, can control the quality of the whole processes of virus extraction and RT-PCR (reverse transcription-polymerase chain reaction) of Hancheng virus detection, and is simple to operate, safe and effective.)

1. An armored RNA standard substance containing Hancheng virus G2 protein genes is characterized in that an expression vector of the armored RNA is a pET32a-CP-G2 plasmid, and the plasmid contains a phage MS2CP protein sequence fragment and a Hancheng virus G2 protein sequence fragment; transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, performing induced expression, performing ultrasonic disruption, centrifuging, collecting precipitate, and obtaining virus-like particles.

2. A preparation method of armored RNA standard substance containing Hancheng virus G2 protein genes is characterized by comprising the following steps:

s1, phage MS2CP protein gene amplification: a pair of specific primers CPP1 and CP P2 are designed according to the CP gene sequence of the bacteriophage MS2, and the primer sequences are shown as follows:

primer CP P1: 5'-GCGGTACCGGGTGGGACCCCTTTCGGGGTCCTG-3', respectively;

primer CP P2: 5'-TTCCAGTAGCGACAGAAGCAAAAGCTTCC-3', respectively;

MS2 phage gene sequence MS2CP is amplified by PCR by taking the MS2 phage sequence fragment plasmid as a vector and using a primer CP P1 and a primer CP P2, and the sequence is shown as SEQ ID NO. 3;

s2, obtaining of expression vector pET32 a-CP: carrying out double enzyme digestion on the PCR amplification product and a prokaryotic expression vector pET-32a by using BamHI and NotI, carrying out electrophoresis on 1.5% agarose gel, recovering a target fragment subjected to enzyme digestion by using a gel recovery kit, and transforming a DH5 alpha strain after connection to obtain an expression vector pET32 a-CP;

s3, obtaining a Hancheng virus G2 protein fragment gene sequence: artificially synthesizing a genomic sequence of the Hancheng virus G2, wherein the sequence is shown as SEQ ID NO. 4, inserting BamHI and Not I enzyme cutting sites into the 5 'end and the 3' end of the sequence respectively, and connecting the sequence to a pBluescript II SK + plasmid vector after enzyme cutting to obtain a target sequence fragment pBSK-G2;

s4, acquisition of expression plasmid pBSK-G2: BamHI and Not I are used for double enzyme digestion of pET32a-CP and pBSK-G2, electrophoresis is carried out on 1.5% agarose gel, a gel recovery kit is used for recovering target fragments, and DH5 alpha strain is transformed after connection, so that the Hancheng virus armored RNA expression vector pET32a-CP-G2 is obtained.

S5, obtaining Hancheng virus armored RNA: transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, performing induced expression, performing ultrasonic disruption, centrifuging, collecting precipitate, and obtaining virus-like particles.

3. The method for preparing the armored RNA standard substance containing the Hancheng virus G2 protein gene according to claim 2, wherein the MS2 phage gene sequence MS2CP in the step S1 comprises the 5' non-coding region sequence of the MS2 phage genome, the mature enzyme protein gene sequence, the capsid protein gene, the packaging site and the replicase gene partial sequence and carries out optimization adjustment on the partial sequence; kpn I and BamHI cleavage sites were inserted into the 5 'end of the primer CP 1 sequence and the 3' end of the primer CP 2 sequence, respectively.

4. The method for preparing the armored RNA standard substance containing the Hancheng virus G2 protein gene according to claim 2, wherein the PCR reaction system in step S1 comprises the following components in the following concentrations: 2 μ L of 10 XPCR Buffer, 1.6 μ L of dNTP of 2.5mMeach, 0.4 μ L of rTaq of 5U/μ L, 0.2 μ L of Mgcl 21.2 μ L, 20pmol/L of primer CP P1 partner primer CPP 21 μ L, DDW 11.8.8 μ L, plasmid pUC18-MS 21 μ L; the reaction conditions were 94 ℃ for 5min, 94 ℃ for 30sec, 52 ℃ for 30sec, 72 ℃ for 1min, and 72 ℃ for 7min in this order.

5. The method for preparing the armored RNA standard substance containing the Hancheng virus G2 protein gene according to claim 2, wherein the enzyme digestion reaction system in the step S2 comprises the following components in the following concentrations: 10 XK Buffer 1 uL, 15 units/. mu.L BamHI 1 uL, 10 units/. mu.L Kpn I1. mu. L, PCR product and pET32a plasmid 1. mu.L DDw 16. mu.L, enzyme digestion 1h at 37 ℃;

the linking system in step S2 included the following components at the following concentrations: ligation MiX 10. mu. L, CP 8. mu. L, pET32a 2. mu.l, 4 ℃ overnight.

6. The method for preparing the armored RNA standard substance containing the Hancheng virus G2 protein gene according to claim 2, wherein the step S4 specifically comprises the following steps: transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, transferring and inoculating 2mL of LB liquid culture medium containing Amp 100 mug/mL at 37 ℃ for overnight culture by positive cloning, transferring and inoculating 2mL of 2 XYT liquid culture medium at a ratio of 1:100, shaking at 37 ℃ and 200rpm for 4 hours, adding 1mM IPTG, inducing for 5 hours, and terminating the culture; and (4) centrifugally collecting thalli, carrying out ultrasonic crushing, and centrifugally taking supernatant to obtain crude virus-like particles.

7. The method for preparing the armored RNA standard substance containing the Hancheng virus G2 protein gene according to claim 2, wherein the enzyme digestion reaction system in the step S4 comprises the following components in the following concentrations: 10 XK Buffer 1 muL, 15 units/muL BamHI 1 muL, 10 units/muL Not I1 muL, 0.1% BSA1 muL, plasmid 10 muL, DDW 6 muL, enzyme digestion at 37 ℃ for 3 h;

the linking system in step S4 included the following components at the following concentrations: ligation MiX 10. mu. L, pET32a-CP 2. mu. L, G28. mu.L was reacted overnight at 4 ℃.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an armored RNA standard substance containing Hancheng virus G2 protein genes and a preparation method thereof.

Background

In recent years, hemorrhagic fever with renal syndrome infected by Hancheng virus is frequently reported and becomes an important public health problem; the Hancheng virus (Seoul virus) belongs to the family Bunyaviridae (Bunyaviridae) the genus Hantaan virus (Hantaan virus). The main host of the Hancheng virus is the brown rat, and in China, the black rat, the yellow corteur and the little rat can carry the Hancheng virus. China is the most seriously harmed country by Hantaan virus, and the number of hemorrhagic fever with renal syndrome accounts for more than 90 percent of the worldwide reported cases. In 1981, hemorrhagic fever epidemic caused by Hancheng virus occurs in Henan and Shanxi junction areas of China. Furthermore, hancheng virus is the only hantavirus that is distributed worldwide, and its presence is found in asia, europe, africa, and north-south america. The hancheng virus carried by the rats of the host animal spreads the virus to humans through feces, urine, saliva or blood into the damaged skin and mucous membranes, respiratory tract and digestive tract of humans, or causes infection by biting humans through fleas, mites and the like. The molecular biology technology represented by the PCR technology has the advantages of rapidness, sensitivity, low cost and the like, and meanwhile, the host epidemic situation of the Hancheng virus can be obtained by performing molecular epidemiological analysis on the detection result by utilizing the PCR detection technology, so that the method is favorable for preventing and controlling the epidemic of the hemorrhagic fever with renal syndrome.

Nucleic acid detection must use quality control products for quality control, and the quality control products of the current RNA virus mainly comprise virus particles, naked RNA fragments and armored RNA. Viral particles include attenuated, inactivated viruses or vaccines. Currently, there is a live attenuated vaccine strain Z37 for hantavirus. However, the virus preparation needs to be carried out in a high-specification biosafety laboratory, and the virus is taken as a quality control standard substance due to the limitation of transportation and storage. At present, various hemorrhagic fever virus detection kits sold in the market at home generally adopt synthetic RNA or DNA, RNA templates are easily degraded by RNase in the environment, DNA templates cannot participate in the nucleic acid extraction process and the reverse transcription process, and the whole detection process cannot be monitored. At present, phage-based embedded nucleic acid virus armour particles have been a hot spot for standard substance development because they are closest to whole virus particles. The Escherichia coli MS2 phage envelope protein and operon RNA sequence have specific interaction, phage gene RNA is packaged into the envelope while the phage shell is assembled, and the phage shell can tolerate RNase, so that the corresponding cDNA sequence of MS2 phage is cloned to a prokaryotic expression vector, inserted into a specific virus sequence, transcribed and translated into phage capsid protein to assemble virus-like particles, and the virus-like particles have RNase resistance and are convenient to store and transport.

Therefore, an armored RNA standard substance containing the Hancheng virus G2 protein gene and a preparation method thereof need to be proposed.

Disclosure of Invention

In order to overcome the defects of the existing quality control sample or standard substance, the invention provides the armored RNA standard substance containing the Hancheng virus G2 protein gene and the preparation method thereof, which have the characteristics of stability, safety, ribonucleic acid resistance and the like and can be used for quality control of Hancheng virus detection.

In order to solve the technical problems, the invention provides the following technical scheme:

the first purpose of the invention is to provide an armored RNA standard substance containing Hancheng virus G2 protein genes, which is characterized in that an expression vector of the armored RNA is a pET32a-CP-G2 plasmid, and the plasmid contains a bacteriophage MS2CP protein sequence fragment and a Hancheng virus G2 protein sequence fragment; transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, performing induced expression, performing ultrasonic disruption, centrifuging, collecting precipitate, and obtaining virus-like particles.

The second purpose of the invention is to provide an armored RNA standard substance containing Hancheng virus G2 protein genes, which is prepared by the following method:

s1, phage MS2CP protein gene amplification: a pair of specific primers CP P1 and CP P2 were designed based on the CP gene sequence of bacteriophage MS2, and the primer sequences are shown below:

primer CP P1: 5'-GCGGTACCGGGTGGGACCCCTTTCGGGGTCCTG-3', respectively;

primer CP P2: 5'-TTCCAGTAGCGACAGAAGCAAAAGCTTCC-3', respectively;

MS2 phage gene sequence MS2CP is amplified by PCR by taking the MS2 phage sequence fragment plasmid as a vector and using a primer CP P1 and a primer CP P2, and the sequence is shown as SEQ ID NO. 3;

s2, obtaining of expression vector pET32 a-CP: carrying out double enzyme digestion on the PCR amplification product and a prokaryotic expression vector pET-32a by using BamHI and Not I, carrying out electrophoresis on 1.5% agarose gel, recovering a target fragment subjected to enzyme digestion by using a gel recovery kit, and transforming a DH5 alpha strain after connection to obtain an expression vector pET32 a-CP;

s3, obtaining a Hancheng virus G2 protein fragment gene sequence: artificially synthesizing a genomic sequence of the Hancheng virus G2, wherein the sequence is shown as SEQ ID NO. 4, inserting BamHI and Not I enzyme cutting sites into the 5 'end and the 3' end of the sequence respectively, and connecting the sequence to a pBluescript II SK + plasmid vector after enzyme cutting to obtain a target sequence fragment pBSK-G2;

s4, acquisition of expression plasmid pBSK-G2: BamHI and Not I are used for double enzyme digestion of pET32a-CP and pBSK-G2, electrophoresis is carried out on 1.5% agarose gel, a gel recovery kit is used for recovering target fragments, and DH5 alpha strain is transformed after connection, so that the Hancheng virus armored RNA expression vector pET32a-CP-G2 is obtained.

S5, obtaining Hancheng virus armored RNA: transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, performing induced expression, performing ultrasonic disruption, centrifuging, collecting precipitate, and obtaining virus-like particles.

As a preferred technical scheme of the invention, the MS2 phage gene sequence MS2CP in the step S1 comprises a 5' non-coding region sequence of the MS2 phage genome, a mature enzyme protein gene sequence, a capsid protein gene, a packaging site and a replicase gene partial sequence and carries out optimization adjustment on the partial sequence; kpn I and BamHI cleavage sites were inserted into the 5 'end of the primer CP 1 sequence and the 3' end of the primer CP 2 sequence, respectively.

As a preferred embodiment of the present invention, the PCR reaction system in step S1 comprises the following components in the following concentrations: 2 μ L of 10 XPCR Buffer, 1.6 μ L of dNTP of 2.5mM each, 0.4 μ L of rTaq of 5U/. mu.L, 21.2 μ L of Mgcl, 20pmol/L of primer CP 1 partner primer CP 21 μ L, DDW 11.8.8 μ L, plasmid pUC18-MS 21 μ L; the reaction conditions were 94 ℃ for 5min, 94 ℃ for 30sec, 52 ℃ for 30sec, 72 ℃ for 1min, and 72 ℃ for 7min in this order.

As a preferred technical solution of the present invention, in step S2, the enzyme digestion reaction system contains the following components at the following concentrations: 10 XK Buffer 1 uL, 15 units/. mu.L BamHI 1 uL, 10 units/. mu.L Kpn I1. mu. L, PCR product and pET32a plasmid 1. mu.L DDw 16. mu.L, enzyme digestion 1h at 37 ℃;

the linking system in step S2 included the following components at the following concentrations: ligation MiX 10. mu. L, CP 8. mu. L, pET32a 2. mu.l, 4 ℃ overnight.

As a preferred technical solution of the present invention, step S4 specifically includes the following steps: transferring the Hancheng virus armored RNA expression vector pET32a-CP-G2 into an escherichia coli prokaryotic expression strain BL21, transferring and inoculating 2mL of LB liquid culture medium containing Amp 100 mug/mL at 37 ℃ for overnight culture by positive cloning, transferring and inoculating 2mL of 2 XYT liquid culture medium at a ratio of 1:100, shaking at 37 ℃ and 200rpm for 4 hours, adding 1mM IPTG, inducing for 5 hours, and terminating the culture; and (4) centrifugally collecting thalli, carrying out ultrasonic crushing, and centrifugally taking supernatant to obtain crude virus-like particles.

As a preferred technical solution of the present invention, in step S4, the enzyme digestion reaction system contains the following components at the following concentrations: 10 XK Buffer 1 muL, 15 units/muL BamHI 1 muL, 10 units/muL Not I1 muL, 0.1% BSA1 muL, plasmid 10 muL, DDW 6 muL, enzyme digestion at 37 ℃ for 3 h;

the linking system in step S4 included the following components at the following concentrations: ligation MiX 10. mu. L, pET32a-CP 2. mu. L, G28. mu.L was reacted overnight at 4 ℃.

The invention has the beneficial effects that: compared with the existing RNA quality control product, the armored RNA has the advantages that the expression vector of the armored RNA is pET32a-CP-G2 plasmid, and the plasmid contains a bacteriophage MS2CP protein sequence fragment and a Hancheng virus G2 protein sequence fragment. The envelope protein of the bacteriophage MS2 has specific interaction with the RNA sequence of an operon, and the bacteriophage genome RNA can be packaged into the envelope in a bacteriophage coat assembly chamber. Therefore, cDNA of specific RNA is cloned at the downstream of the cDNA of the gene sequence of the MS2 bacteriophage envelope protein, and partial bacteriophage genome of the inserted RNA sequence is packaged into the envelope when the envelope protein of the bacteriophage is assembled into the envelope, thereby forming the bacteriophage virus-like particle. Compared with the existing RNA quality control products, the Hancheng virus armored RNA standard substance prepared by the invention has the characteristics of stability, no biological safety hazard, RNase degradation resistance and the like, is convenient to store, transport and use, can control the quality of the whole processes of virus extraction and RT-PCR (reverse transcription-polymerase chain reaction) of Hancheng virus detection, and is simple to operate, safe and effective.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.