CN108103033A - A kind of fixed point remodeling method of rna virus cdna group - Google Patents
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- CN108103033A CN108103033A CN201711287804.4A CN201711287804A CN108103033A CN 108103033 A CN108103033 A CN 108103033A CN 201711287804 A CN201711287804 A CN 201711287804A CN 108103033 A CN108103033 A CN 108103033A
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Abstract
The present invention relates to biotechnology technical field, especially a kind of fixed point remodeling method of rna virus cdna group comprises the following steps：S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and the protein nucleic acid enzyme system with fixed point cutting RNA activity；S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；S3, structure sick cell；S4, transfectional cell；S5, progeny virus harvest liquid is collected；S6, progeny virus is isolated.The present invention can efficiently realize the fixed point transformation of rna virus cdna group, and can quickly and easily screen and obtain the recombinant virus with specific mutation type.
Description
Technical field
The present invention relates to biotechnology technical field more particularly to a kind of fixed point remodeling methods of rna virus cdna group.
Background technology
The inhereditary material that RNA virus is is made of (RNAribonucleicacid) ribonucleic acid, and usual nucleic acid is single
(ssRNAsingle-strandedRNA) of chain also has the RNA diseases that (dsRNAdouble-strandedRNA) of double-strand is single-stranded
Poison root can be divided into according to their translational meaning just translates, bears the RNA virus translated with two-way translation, the RNA virus just translated and mRNA phases
It seemingly, can be directly by host cell translation into protein；Negative RNA virus of translating then needs effect by RNase, using itself as mould
Plate compiles out the RNA with provirus phase antisense, translates into protein again with this RNA afterwards.
At present for the rite-directed mutagenesis of DNA virus genome, there are mainly two types of methods, and a kind of method is by means of restricted
The toolenzymes such as restriction endonuclease carry out viral genome extracellular genetic recombination operation, but for larger viral genome, very
Difficulty finds the single restriction enzyme site that can be used.Another method is by means of intracellular homologous recombination system, is led to
The homologous sequence that transfection is similar to viral genome into virus infected cell is crossed, completes to determine specific region genome sequence
Point editor since this homologous recombination efficiency is extremely low, is not appropriate for high-throughput virus vaccine strain screening, in order to overcome this
Kind is difficult, generally requires the screening for importing exogenous resistant gene to virus and being used for recombinant virus, this to operate not only time-consuming takes
Power, and it is not appropriate on biological safety the requirement of vaccine strain.How simply, accurately and accurately to large-scale DNA virus base
It is still a technical bottleneck in vaccine development because group carries out fixed point editor.
The content of the invention
The purpose of the present invention is to solve shortcoming in the prior art, and a kind of rna virus cdna group proposed
Fixed point remodeling method.
To achieve these goals, present invention employs following technical solutions：
A kind of fixed point remodeling method of rna virus cdna group is designed, is comprised the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point
Cut the protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is by weight percent
Than for 5-25% ox meat extract powder, 40-60% caseinhydrolysates, the starch of 2-16% is added in after the agar mixing of 15-36% and steamed
Distilled water hydrolyzes, and is uniformly mixed by volume for 8-22: 1 with human red blood cells liquid, obtains cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbed, then suck virus liquid, cultivated extremely
The complete lesion of cell, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system is obtained using S1 and S2 and homologous sequence is total to
Same transfectional cell, obtains transfectional cell, wherein, it is 1 to control the ratio between the quality of cell quantity and express nucleic acid enzyme system
×105:0.5-3μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell is broken
Piece, gained supernatant are progeny virus harvest liquid；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid
Progeny virus is isolated in sequencing identification；Complete the fixed point transformation of rna virus cdna group.
Preferably, the number of insetion sequence is two.
Preferably, transfectional cell is obtained after transfecting 12-36h in S4.
Preferably, the cultivation time of cell is 15-20h in S3.
Preferably, human red blood cells liquid is that remaining red blood cell is in sterile flask after extracting blood plasma by people's venous blood, with sterile life
Salt water washing is managed, is stood, abandoning supernatant forms.
A kind of fixed point remodeling method of rna virus cdna group proposed by the present invention, advantageous effect are：The present invention can be high
The fixed point transformation of the realization rna virus cdna group of effect, and can quickly and easily screen and obtain the weight with specific mutation type
Group virus.
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, instead of all the embodiments.
Embodiment 1
A kind of fixed point remodeling method of rna virus cdna group, comprises the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point
Cut the protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is by weight percent
Than for 5% N of meat extract powder, 40% caseinhydrolysate, 2% starch, add in distilled water hydrolysis after 15% agar mixing and
Into being uniformly mixed by volume for 8: 1 with human red blood cells liquid, obtain cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbed, then suck virus liquid, cultivated extremely
The complete lesion of cell, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system is obtained using S1 and S2 and homologous sequence is total to
Same transfectional cell, obtains transfectional cell, wherein, it is 1 to control the ratio between the quality of cell quantity and express nucleic acid enzyme system
×105:0.5μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell is broken
Piece, gained supernatant are progeny virus harvest liquid；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid
Progeny virus is isolated in sequencing identification；Complete the fixed point transformation of rna virus cdna group.
The number of insetion sequence is two.
Transfectional cell is obtained after transfecting 12h in S4.
The cultivation time of cell is 15h in S3.
Human red blood cells liquid is that remaining red blood cell in sterile flask, is washed with sterile saline after extracting blood plasma by people's venous blood
It washs, stands, abandoning supernatant forms.
Embodiment 2
A kind of fixed point remodeling method of rna virus cdna group, comprises the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point
Cut the protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is by weight percent
Than for 6% N of meat extract powder, 45% caseinhydrolysate, 3% starch, add in distilled water hydrolysis after 18% agar mixing and
Into being uniformly mixed by volume for 10: 1 with human red blood cells liquid, obtain cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbed, then suck virus liquid, cultivated extremely
The complete lesion of cell, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system is obtained using S1 and S2 and homologous sequence is total to
Same transfectional cell, obtains transfectional cell, wherein, it is 1 to control the ratio between the quality of cell quantity and express nucleic acid enzyme system
×105:0.6μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell is broken
Piece, gained supernatant are progeny virus harvest liquid；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid
Progeny virus is isolated in sequencing identification；Complete the fixed point transformation of rna virus cdna group.
The number of insetion sequence is two.
Transfectional cell is obtained after transfecting 14h in S4.
The cultivation time of cell is 16h in S3.
Human red blood cells liquid is that remaining red blood cell in sterile flask, is washed with sterile saline after extracting blood plasma by people's venous blood
It washs, stands, abandoning supernatant forms.
Embodiment 3
A kind of fixed point remodeling method of rna virus cdna group, comprises the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point
Cut the protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is by weight percent
Than for 15% N of meat extract powder, 55% caseinhydrolysate, 10% starch, add in distilled water hydrolysis after 25% agar mixing and
Into being uniformly mixed by volume for 20: 1 with human red blood cells liquid, obtain cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbed, then suck virus liquid, cultivated extremely
The complete lesion of cell, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system is obtained using S1 and S2 and homologous sequence is total to
Same transfectional cell, obtains transfectional cell, wherein, it is 1 to control the ratio between the quality of cell quantity and express nucleic acid enzyme system
×105:2μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell is broken
Piece, gained supernatant are progeny virus harvest liquid；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid
Progeny virus is isolated in sequencing identification；Complete the fixed point transformation of rna virus cdna group.
The number of insetion sequence is two.
Transfectional cell is obtained after transfecting 25h in S4.
The cultivation time of cell is 18h in S3.
Human red blood cells liquid is that remaining red blood cell in sterile flask, is washed with sterile saline after extracting blood plasma by people's venous blood
It washs, stands, abandoning supernatant forms.
Embodiment 4
A kind of fixed point remodeling method of rna virus cdna group, comprises the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point
Cut the protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is by weight percent
Than for 25% N of meat extract powder, 60% caseinhydrolysate, 16% starch, add in distilled water hydrolysis after 36% agar mixing and
Into being uniformly mixed by volume for 22: 1 with human red blood cells liquid, obtain cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbed, then suck virus liquid, cultivated extremely
The complete lesion of cell, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system is obtained using S1 and S2 and homologous sequence is total to
Same transfectional cell, obtains transfectional cell, wherein, it is 1 to control the ratio between the quality of cell quantity and express nucleic acid enzyme system
×105:3μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell is broken
Piece, gained supernatant are progeny virus harvest liquid；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid
Progeny virus is isolated in sequencing identification；Complete the fixed point transformation of rna virus cdna group.
The number of insetion sequence is two.
Transfectional cell is obtained after transfecting 36h in S4.
The cultivation time of cell is 20h in S3.
Human red blood cells liquid is that remaining red blood cell in sterile flask, is washed with sterile saline after extracting blood plasma by people's venous blood
It washs, stands, abandoning supernatant forms.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. a kind of fixed point remodeling method of rna virus cdna group, which is characterized in that comprise the following steps：
S1, the nucleic acid enzyme system of structure fixed point cutting：Build at least one molecule comprising guide function and with fixed point cutting
The protein nucleic acid enzyme system of RNA activity；
S2, structure include the homologous sequence of specific mutations, missing or insetion sequence；
S3, structure sick cell：
The culture of A1, cell：After choosing caseinhydrolysate agar sterilization treatment, caseinhydrolysate agar is to be by weight percent
5-25% ox meat extract powder, 40-60% caseinhydrolysates, the starch of 2-16% add in distilled water after the agar mixing of 15-36%
It hydrolyzes, is uniformly mixed by volume for 8-22: 1 with human red blood cells liquid, obtains cell culture fluid；
A2, virus liquid is added in the culture plate for being contained with cell culture fluid, adsorbs, then suck virus liquid, cultivate to cell
Complete lesion, obtains sick cell；
S4, transfectional cell：The sick cell of previous step is collected, nucleic acid enzyme system and homologous sequence is obtained using S1 and S2 and turns jointly
Contaminate cell, obtain transfectional cell, wherein, control ratio between the quality of cell quantity and express nucleic acid enzyme system be 1 ×
105:0.5-3μg；
S5, sick cell and/or supernatant obtained in the previous step are collected, after freeze thawing or supersound process, centrifugation removal cell fragment, institute
It is progeny virus harvest liquid to obtain supernatant；
S6, from progeny virus harvest liquid obtained in the previous step, picking derive from monoclonal progeny virus, pass through nucleic acid sequencing
Identification, isolates progeny virus；Complete the fixed point transformation of rna virus cdna group.
2. a kind of fixed point remodeling method of rna virus cdna group according to claim 1, it is characterised in that：Insetion sequence
Number be two.
3. a kind of fixed point remodeling method of rna virus cdna group according to claim 1, it is characterised in that：It is transfected in S4
Transfectional cell is obtained after 12-36h.
4. a kind of fixed point remodeling method of rna virus cdna group according to claim 1, it is characterised in that：Cell in S3
The cultivation time be 15-20h.
5. a kind of fixed point remodeling method of rna virus cdna group according to claim 1, it is characterised in that：Human red blood cells
Liquid is that remaining red blood cell in sterile flask, is washed with sterile saline after extracting blood plasma by people's venous blood, is stood, supernatant discarding
Liquid forms.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1224061A (en) * | 1998-01-21 | 1999-07-28 | 鞠秋艳 | Method for preparing human red blood cell culture medium |
| CN103397018A (en) * | 2013-08-20 | 2013-11-20 | 中国医学科学院医学生物学研究所 | Site-directed modification method for DNA viral genome |
| CN103757053A (en) * | 2014-01-28 | 2014-04-30 | 中国医学科学院医学生物学研究所 | Site-specific modification and screening method for specific DNA (deoxyribonucleic acid) viral genome |
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- 2017-12-07 CN CN201711287804.4A patent/CN108103033A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1224061A (en) * | 1998-01-21 | 1999-07-28 | 鞠秋艳 | Method for preparing human red blood cell culture medium |
| CN103397018A (en) * | 2013-08-20 | 2013-11-20 | 中国医学科学院医学生物学研究所 | Site-directed modification method for DNA viral genome |
| CN103757053A (en) * | 2014-01-28 | 2014-04-30 | 中国医学科学院医学生物学研究所 | Site-specific modification and screening method for specific DNA (deoxyribonucleic acid) viral genome |
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Application publication date: 20180601 |