阅读说明：本技术 制备慢病毒颗粒包装ebovrna作为ebov核酸检测阳性参考品的方法 (Method for preparing lentiviral particle packaging EBOVRNA as EBOV nucleic acid detection positive reference substance ) 是由 危宏平 余军平 熊进 于 2019-10-15 设计创作，主要内容包括：本发明公开了制备慢病毒颗粒包装EBOV RNA作为EBOV核酸检测阳性参考品的方法,制备方法将EBOV的三个基因NP、GP和L基因的部分片段串联后整合到慢病毒的表达载体中,将构建的慢病毒表达质粒与包装质粒分别在工程大肠杆菌菌株中扩增,得到大量的质粒,再将构建的表达质粒和包装质粒共同转染至HEK293T细胞中,经细胞表达后得到大量的包装了EBOV的RNA基因的慢病毒颗粒。本发明涉及基因工程技术领域。该制备慢病毒颗粒包装EBOV RNA作为EBOV核酸检测阳性参考品的方法,得到的颗粒作为EBOV核酸检测的阳性参考品,具有很好模拟实际样本中从病毒核酸提取到核酸检测的整个过程的优点,将EBOV的假病毒颗粒通过冻干保护剂进行冻干后得到的冻干粉,非常稳定。(The invention discloses a method for preparing a lentiviral particle packaging EBOV RNA as an EBOV nucleic acid detection positive reference product, which comprises the steps of connecting partial fragments of NP, GP and L genes of EBOV in series, integrating the partial fragments into an expression vector of lentivirus, respectively amplifying constructed lentivirus expression plasmids and packaging plasmids in engineering escherichia coli strains to obtain a large number of plasmids, then co-transfecting the constructed expression plasmids and the packaging plasmids into HEK293T cells, and obtaining a large number of lentiviral particles packaging the EBOV RNA genes after cell expression. The invention relates to the technical field of genetic engineering. According to the method for preparing the EBOV RNA packaged by the lentivirus particles as the EBOV nucleic acid detection positive reference substance, the obtained particles as the EBOV nucleic acid detection positive reference substance have the advantage of well simulating the whole process of extracting the virus nucleic acid from an actual sample to detect the nucleic acid, and the freeze-dried powder obtained by freeze-drying the EBOV pseudovirus particles through the freeze-drying protective agent is very stable.)

1. The method for preparing the EBOVRNA packaged by the lentivirus particles as the EBOV nucleic acid detection positive reference substance is characterized by comprising the following steps of: the method specifically comprises the following steps:

s1, cutting the synthesized EBOV gene with XbaI and BamHI by XbaI and BamHI, and cutting the lentivirus expression plasmid by XbaI and BamHI;

s2, connecting the nucleotide fragment of the EBOV gene obtained in the step S1 with a lentiviral expression vector by using T4 ligase to construct a recombinant lentiviral expression vector containing the EBOV gene;

s3, respectively transforming the constructed recombinant plasmids and the three packaging plasmids of the lentivirus into escherichia coli, amplifying and extracting corresponding plasmids;

s4, transfecting the 4 plasmids obtained in the step S3 to cells, and expressing and packaging pseudovirus particles to obtain pseudoviruses packaged with EBOV RNA;

s5, collecting cell culture solution supernatant obtained in the step S4, and filtering to obtain a crude product;

s6, precipitating the filtrate obtained in the step S5 by PEG8000, collecting the precipitate and re-suspending the precipitate into buffer PBS;

s7, dialyzing the solution obtained in the step S6 by a dialysis bag to remove PEG 8000;

s8, treating the solution obtained in the step S7 with DNaseI, superionizing the solution, precipitating the solution in PBS, superionizing the solution once again, and precipitating the solution in PBS;

s9, adding the solution obtained in the step S8 into a freeze-drying protective agent, and freeze-drying to obtain the EBOV RNA-packaged pseudovirus freeze-dried powder.

2. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the lentiviral expression vector used in step S1 was pEB-copGFP (T2A) PURO.

3. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the three packaging plasmids in step S3 were VSV-G, PLP1 and PLP2, respectively.

4. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the Escherichia coli in the step S3 is DH5 alpha.

5. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the transfected cells in the step S4 were HEK293T cells.

6. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the specific steps of transforming the lentiviral expression vector plasmid together with 3 packaging plasmids into HEK293T in step S4 are as follows:

t1, 100-200 mu L of serum-free cell culture medium and 30 mu L of P3000 in lipofectamine3000 cell transfection kit^TMThe reagent, 3-15 mu g of PLP1, 3-15 mu g of PLP2, 3-15 mu g of VSVG and 3-15 mu g of pEB-copGFP (T2A) PURO-EBOV are subjected to constant volume to 650 mu L by using a serum-free cell culture medium and are gently mixed;

t2, taking 575 mu L of serum-free cell culture medium, diluting 75 mu L of lipofectamine3000 reagent, and gently mixing the reagent after the reagent is added;

t3, adding 650 mu L of the mixed solution in the step T1 into 650 mu L of the mixed solution in the step T2, gently mixing the mixed solution uniformly, and incubating the mixed solution at room temperature for 5 min;

t4, completely sucking the culture medium in the cell culture plate by using a pipette, washing the cells by using 1-5mL of D-Hanks buffer solution, and uniformly adding the liposome solution in the step T3 after finishing the incubation to 25cm²Incubating the cells in the cell culture plate at 37 ℃ for 15 min;

and adding 5mL of cell culture solution containing 10% fetal calf serum into the T5 cell culture disc, culturing in a cell culture box containing 5% carbon dioxide at 37 ℃, replacing fresh cell culture solution after 6h, collecting supernatant twice within 24-120 h, and combining the supernatants collected twice.

7. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the specific steps of PEG8000 precipitation of lentivirus particles in the step S6 are as follows: adding solid NaCl into the filtered cell culture solution supernatant to a concentration of 1M, adding solid PEG8000 to a final concentration of 10% (w/v) after dissolution, fully shaking for dissolution, standing overnight in a four-degree refrigerator to precipitate lentiviral particles, centrifuging at 10,000rpm at 4 ℃ for 15min, discarding the supernatant, then resuspending the precipitate with PBS buffer, and transferring to a clean 2mL ep tube.

8. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the method for removing PEG8000 used in step S7 is a dialysis method, and low temperature dialysis is carried out for 6 hours with a 7K dialysis bag, and the solution is changed 3 times.

9. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the DNaseI treatment concentration, the DNaseI treatment temperature and the DNaseI treatment time in the step S8 are respectively 0.5-2 mg/mL, 37 ℃ and 1h, the DNaseI removal method in the step S8 is that the DNaseI is removed at 20,000-40,000 rpm for 2-4 h, the DNaseI is repeatedly removed once, the precipitate is collected, dissolved by PBS, and split-packaged and frozen.

10. The method for preparing an ebovr packaged lentiviral EBOVRNA as a positive reference for EBOV nucleic acid detection according to claim 1, wherein: the components of the freeze-drying protective agent in the step S9 are 10-30% of trehalose and 1-3% of BSA respectively, and the freeze-drying protective procedure is as follows:

A1、Freeze-30℃,1h 30min；

A2、Freeze-30℃,2h；

A3、Condenser preparation,15min；

A4、Chamber vacuum,400μbar；

A5、Primary drying,-40℃,200μbar,1h；

A6、Primary drying,-40℃,200μbar,17h；

A7、Secondary drying 4℃,1h；

A8、Secondary drying 4℃,4h；

A9、End of cycle。

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a preparation method of a positive reference substance for EBOV nucleic acid detection by using RNA of partial fragments of NP, GP and L genes of three genes of slow virus particle packaging Ebola virus (EBOV), and a method for stably storing the positive reference substance.

Background

Ebola virus (EBOV) has extremely high lethality and infectivity, is a virulent pathogen with extremely high global concern, the commonly used detection method is mainly RT-PCR, a corresponding kit must be matched with a positive reference substance, the positive reference substance of the nucleic acid detection kit of the EBOV is generally plasmid DNA, reverse transcription RNA or inactivated virus, and the positive reference substances have the following different defects at present:

1. if the inactivated virus needs four-level biological safety laboratory preparation, and the safety of the inactivated virus cannot be guaranteed;

2. the reverse transcription RNA is unstable in property, easy to degrade and difficult to store;

3. and plasmid DNA as a universal positive template cannot simulate the whole process of extracting a sample from RNA to nucleic acid amplification detection, and the simulation is insufficient.

These reasons all lead to inaccurate detection results and even false negative results, so that how to construct a positive reference substance which can simulate the whole process from RNA extraction to nucleic acid amplification detection steps of the virus as a positive reference of the kit is an urgent problem to be solved.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for preparing an EBOV RNA packaged by lentivirus particles as an EBOV nucleic acid detection positive reference substance, the EBOV nucleic acid positive reference substance can completely simulate the whole process of virus RNA extraction and nucleic acid detection, has high stability, can be used for positive reference of an EBOV nucleic acid detection kit, and can obtain the lentivirus particles packaged with the EBOV RNA with high stability and high copy number by using the preparation method established by the invention.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: the method for preparing the lentiviral particle packaging EBOVRNA as the EBOV nucleic acid detection positive reference substance specifically comprises the following steps:

s1, cutting the synthesized EBOV gene with XbaI and BamHI by XbaI and BamHI, and cutting the lentivirus expression plasmid by XbaI and BamHI;

s2, connecting the nucleotide fragment of the EBOV gene obtained in the step S1 with a lentiviral expression vector by using T4 ligase to construct a recombinant lentiviral expression vector containing the EBOV gene;

s3, respectively transforming the constructed recombinant plasmids and the three packaging plasmids of the lentivirus into escherichia coli, amplifying and extracting corresponding plasmids;

s4, transfecting the 4 plasmids obtained in the step S3 to cells, and expressing and packaging pseudovirus particles to obtain pseudoviruses packaged with EBOV RNA;

s5, collecting cell culture solution supernatant obtained in the step S4, and filtering to obtain a crude product;

s6, precipitating the filtrate obtained in the step S5 by PEG8000, collecting the precipitate and re-suspending the precipitate into buffer PBS;

s7, dialyzing the solution obtained in the step S6 by a dialysis bag to remove PEG 8000;

s8, treating the solution obtained in the step S7 with DNaseI, superionizing the solution, precipitating the solution in PBS, superionizing the solution once again, and precipitating the solution in PBS;

s9, adding the solution obtained in the step S8 into a freeze-drying protective agent, and freeze-drying to obtain the EBOV RNA-packaged pseudovirus freeze-dried powder.

Preferably, the lentiviral expression vector used in step S1 is pEB-copGFP (T2A) PURO.

Preferably, the three packaging plasmids in step S3 are VSV-G, PLP1 and PLP2, respectively.

Preferably, the escherichia coli in the step S3 is DH5 α.

Preferably, the transfected cells in the step S4 are HEK293T cells.

Preferably, the specific steps of transforming the lentiviral expression vector plasmid together with 3 packaging plasmids into HEK293T in step S4 are as follows:

t1, 100-200 mu L of serum-free cell culture medium (DMEM) and 30 mu L of P3000 in lipofectamine3000 cell transfection kit^TMThe reagent, 3-15 mu g of PLP1, 3-15 mu g of PLP2, 3-15 mu g of VSVG and 3-15 mu g of pEB-copGFP (T2A) PURO-EBOV are subjected to constant volume to 650 mu L by using a serum-free cell culture medium and are gently mixed;

t2, taking 575 mu L of serum-free cell culture medium, diluting 75 mu L of lipofectamine3000 reagent, and gently mixing the reagent after the reagent is added;

t3, adding 650 mu L of the mixed solution in the step T1 into 650 mu L of the mixed solution in the step T2, gently mixing the mixed solution uniformly, and incubating the mixed solution at room temperature for 5 min;

t4, completely sucking the culture medium in the cell culture plate by using a pipette, washing the cells by using 1-5mL of D-Hanks buffer solution, and uniformly adding the liposome solution in the step T3 after finishing the incubation to 25cm²Incubating the cells in the cell culture plate at 37 ℃ for 15 min;

and adding 5mL of cell culture solution containing 10% fetal calf serum into the T5 cell culture disc, culturing in a cell culture box containing 5% carbon dioxide at 37 ℃, replacing fresh cell culture solution after 6h, collecting supernatant twice within 24-120 h, and combining the supernatants collected twice.

Preferably, the PEG8000 precipitating lentiviral particles in step S6 specifically comprises the following steps: adding solid NaCl into the filtered cell culture solution supernatant to a concentration of 1M, adding solid PEG8000 to a final concentration of 10% (w/v) after dissolution, fully shaking for dissolution, standing overnight in a four-degree refrigerator to precipitate lentiviral particles, centrifuging at 10,000rpm at 4 ℃ for 15min, discarding the supernatant, then resuspending the precipitate with PBS buffer, and transferring to a clean 2mL ep tube.

Preferably, the method for removing PEG8000 in step S7 is a dialysis method, which comprises dialyzing with 7K dialysis bag at low temperature for 6 hr and changing the solution for 3 times.

Preferably, the DNaseI treatment concentration, temperature and time in the step S8 are respectively 0.5-2 mg/mL, 37 ℃ and 1h, the DNaseI removal method in the step S8 is that the DNaseI is removed at 20,000-40,000 rpm for 2-4 h, the superseparation is repeated once, the precipitate is collected, dissolved by PBS, and split and frozen.

Preferably, the components of the lyoprotectant in step S9 are 10-30% of trehalose and 1-3% of BSA, respectively, and the lyoprotectant includes:

A1、Freeze-30℃,1h 30min；

A2、Freeze-30℃,2h；

A3、Condenser preparation,15min；

A4、Chamber vacuum,400μbar；

A5、Primary drying,-40℃,200μbar,1h；

A6、Primary drying,-40℃,200μbar,17h；

A7、Secondary drying 4℃,1h；

A8、Secondary drying 4℃,4h；

A9、End of cycle。

preferably, the nucleotide sequence of cDNA corresponding to EBOV RNA packaged by the lentivirus particles is shown as SEQ ID No. 1.

(III) advantageous effects

The invention provides a method for preparing lentiviral particle packaging EBOVRNA as an EBOV nucleic acid detection positive reference substance. Compared with the prior art, the method has the following beneficial effects:

(1) according to the method for preparing the lentiviral-particle-packaged EBOVRNA as the EBOV nucleic acid detection positive reference substance, the EBOV RNA reference substance is obtained through partial fragment RNA sequences of NP, GP and L genes of the lentiviral-packaged EBOV genes, and the reference substance can truly simulate the whole process of extracting RNA from a sample to nucleic acid detection in the EBOV detection process.

(2) According to the method for preparing the EBOVRNA packaged by the lentivirus particles as the EBOV nucleic acid detection positive reference substance, the EBOV RNA nucleic acid reference substance is provided, and the third generation lentivirus packaging system is adopted, so that the safety of the lentivirus obtained by the system is high, and the reference substance for nucleic acid detection does not need to be used in a biological safety laboratory.

(3) According to the method for preparing the EBOV RNA packaged by the lentiviral particles as the EBOV nucleic acid detection positive reference substance, the EBOV RNA packaged lentiviral particles obtained by the freeze-drying process have high stability, and the EBOV RNA packaged lentiviral particles as the EBOV nucleic acid detection positive reference substance do not need low-temperature transportation or low-temperature storage, so that the use cost is greatly saved.

Drawings

FIG. 1 is a TEM image of an EBOV RNA fragment-packaged lentiviral particle prepared in example 3 of the present invention;

FIG. 2 is a graph of a typical qPCR assay in example 3 of the present invention;

FIG. 3 is a diagram showing the detection result of optimizing ddPCR annealing temperature in example 3 of the present invention;

FIG. 4 is a graph showing the results of uniformity measurements on lyophilized tubes of lentiviral particles packaged with EBOV RNA fragments in example 5 of the present invention;

FIG. 5 is a qPCR result graph of EBOV RNA fragment-packaged lentivirus particle lyophilized powder of example 5 of the present invention after two and four weeks at different temperatures;

FIG. 6 is a graph showing the results of ddPCR of the EBOV RNA fragment-packaged lentivirus particle lyophilized powder of example 5 of the present invention placed at different temperatures for two and four weeks.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples are shown to illustrate certain embodiments of the invention in detail, and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Specifically, the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as molecular cloning, described by Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

The commonly used positive reference substance (plasmid DNA, reverse transcription RNA or inactivated virus) for detecting the nucleic acid of the EBOV has the defects of insufficient simulation, instability or potential safety hazard and the like. Therefore, the invention provides a preparation method of the safe and stable EBOV nucleic acid reference substance, which uses the slow virus particles to wrap the EBOV RNA gene, can truly simulate the whole process from RNA extraction to detection, has good stability and high safety, and has good application prospect in the nucleic acid detection kit of the EBOV.

The EBOV nucleic acid reference substance is prepared by packaging partial segment RNA of NP, GP and L genes of EBOV in lentiviral particles, and the sequences of the NP, GP and L genes of the EBOV tandem of the nucleic acid reference substance are shown as SEQ ID No. 1.

In general, the EBOV sequences obtained by sequence combination and EBOVRNA-packaged pseudoviral particles obtained by vector packaging all obtain corresponding positive reference substances based on similar principles, and therefore, the pseudoviral particles packaged with viral EBOV RNA obtained by the above method have similar functions and applications and are also included in the scope of the present invention.

Referring to fig. 1-6, the embodiment of the present invention provides five technical solutions: the method for preparing the lentiviral particle packaging EBOVRNA as the EBOV nucleic acid detection positive reference substance specifically comprises the following embodiments: