CN117025722A - Construction method of SPA6 gene - Google Patents
Construction method of SPA6 gene Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 209
- 238000010276 construction Methods 0.000 title claims abstract description 7
- 239000012634 fragment Substances 0.000 claims abstract description 79
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- 230000000295 complement effect Effects 0.000 claims description 6
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- 239000003550 marker Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
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- 238000003776 cleavage reaction Methods 0.000 description 2
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- 230000005526 G1 to G0 transition Effects 0.000 description 1
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- 241000700605 Viruses Species 0.000 description 1
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Abstract
The application discloses a construction method of an SPA6 gene, which belongs to the field of biological genetic engineering. The method comprises the following steps: designing a vector and a plurality of mutation primers; the method comprises the steps that a part of mutation primers and a carrier realize that the carrier mutates at a plurality of mutation sites through a PCR technology, a plurality of groups of gene fragments are obtained, and the plurality of groups of gene fragments are recombined to construct a complete gene sequence; extracting a first gene fragment on the complete gene sequence and a second gene fragment on a vector by using an enzyme digestion technology; constructing plasmids from the first gene fragment and the second gene fragment; performing secondary mutation on the rest mutation primers and the plasmid containing the preset gene fragment by a PCR technology to construct a gene fragment containing the target gene; obtaining a target gene from the gene fragment containing the target gene by using an enzyme digestion technology, and obtaining a third gene fragment on a plasmid; and constructing the target gene and the third gene fragment into DomainA-6.
Description
Technical Field
The application relates to the field of biological genetic engineering, in particular to a construction method of an SPA6 gene.
Background
Contaminants need to be removed during the production of protein molecules to ensure the purity of the protein product. Such contaminants include non-target biomolecules or microorganisms such as proteins, carbohydrates, lipids, bacteria, viruses, and the like. These contaminants are typically removed from the matrix after elution of the desired product in order to regenerate the matrix prior to subsequent use. Such removal typically includes what is known as a cleaning-in-place (CIP) process, wherein a reagent is used that is capable of eluting contaminants from the stationary phase. The most widely used such reagents at present are NaOH, the concentration of which may vary from 0.1M to 1M depending on the degree of contamination and the nature of the substrate. NaOH is an effective CIP reagent that can achieve multi-pathway reduction of contaminants such as microorganisms, proteins, lipids, nucleic acids, and the like. Another advantage of NaOH is that it can be easily removed without any further treatment. However, this solution requires exposing the substrate to extreme alkaline conditions at a pH exceeding 13, and requires a gene with high alkaline resistance for this purpose.
Disclosure of Invention
The summary of the application is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. The summary of the application is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
In order to solve the technical problems mentioned in the background section above, some embodiments of the present application provide a method for constructing SPA6 gene, comprising the steps of:
designing a carrier and a plurality of mutation primers, and preserving for later use;
the method comprises the steps that a part of mutation primers and a carrier realize that the carrier mutates at a plurality of mutation sites through a PCR technology, a plurality of groups of gene fragments are obtained, and the plurality of groups of gene fragments are recombined to construct a complete gene sequence;
extracting a first gene fragment on the complete gene sequence and a second gene fragment on a vector by using an enzyme digestion technology; and connecting the first gene segment with the second gene segment T4 to construct a plasmid containing a preset gene segment;
performing secondary mutation on the rest mutation primers and the plasmid containing the preset gene fragment by a PCR technology to construct a gene fragment containing the target gene;
obtaining a target gene from the gene fragment containing the target gene by using an enzyme digestion technology, and obtaining a third gene fragment on a plasmid; and connecting the target gene and the third gene fragment through T4 to construct a DomainA-6 containing the target gene;
the sequence of the DomainA-6 gene is shown as SEQ ID NO. 1.
Further, the method comprises the steps of,
the sequence of the plasmid gene containing the preset gene fragment is shown as SEQ ID NO. 2;
the sequence of the target gene is shown as SEQ ID NO. 3;
the sequence of the third gene segment gene is shown as SEQ ID NO. 4.
Further, the method comprises the steps of,
the plurality of mutation primers are:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'、DomainAF4'、DomainAR4'。
further, the method comprises the steps of,
the partial mutation primer comprises the following components:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'。
further, the method comprises the steps of,
the method comprises the steps of carrying out mutation on a plurality of mutation sites by a part of mutation primers and a carrier through a PCR technology, obtaining a plurality of groups of gene fragments, recombining the plurality of groups of gene fragments, and constructing a complete gene sequence, wherein the steps comprise:
carrying out PCR on the DomainAF1', single2-1R, single2-2F, single-2R', single2-3F ', domainAR3' and the vector to realize that the vector is mutated at a plurality of mutation sites and three groups of gene fragments are obtained;
overlapping PCR is carried out on the DomainAF1', the DomainAR3' and the three groups of gene fragments, so that a complete gene sequence is constructed.
Further, the method comprises the steps of,
single2-1R and single2-2R' each have bases partially complementary to each other;
both single2-2F and single2-3F' have bases that are partially complementary to each other.
Further, the method comprises the steps of,
the vector is a single-repeat protein A Domain A SPA 11 protein expression precursor.
Further, the method comprises the steps of,
the sequence of the DomainAF1' gene is shown as SEQ ID NO. 5;
single2-1R, the sequence of the gene is shown as SEQ ID NO. 6;
single2-2F, the sequence of the gene is shown as SEQ ID NO. 7;
single2-2R' gene sequence is shown in SEQ ID NO. 8;
single2-3F', the sequence of the gene is shown as SEQ ID NO. 9;
the sequence of the DomainAR3' gene is shown as SEQ ID NO. 10;
the sequence of the DomainAF4' gene is shown as SEQ ID NO. 11;
the sequence of the DomainAR4' gene is shown in SEQ ID NO. 12.
The application has the beneficial effects that: a construction method of SPA6 gene is provided. The SPA6 gene of the application takes the single repeated protein A Domain A SPA 11 protein expression precursor as a template, and adopts designed mutation primer to carry out rapid point mutation PCR amplification mutation plasmid, and the matrix alkali resistance of the obtained SPA6 gene is very high.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application, are incorporated in and constitute a part of this specification. The drawings and their description are illustrative of the application and are not to be construed as unduly limiting the application.
In addition, the same or similar reference numerals denote the same or similar elements throughout the drawings. It should be understood that the figures are schematic and that elements and components are not necessarily drawn to scale.
In the drawings:
FIG. 1 shows the results of double cleavage by overlapping PCR < XbaI, salI >. Line1 is Marker, line2 is enzyme cutting running glue result.
FIG. 2 shows the results of double cleavage of the vector and the single mutant target gene < SalI, bamHI >. Line1 is the result of double enzyme digestion and gel running of the vector < SalI, bamHI >, line2 is Marker, and Line3 is the result of double enzyme digestion and gel running of the single mutation target gene < SalI, bamHI >.
FIG. 3 shows SPA6 protein gel. Line1 is Marker and Line2 is SPA6 protein.
FIG. 4 shows a bar graph of activity change for 24 hour 1M NaOH treatment of SPA6 and control r-SPA.
Detailed Description
Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the drawings and embodiments of the present disclosure are for illustration purposes only and are not intended to limit the scope of the present disclosure.
It should be noted that, for convenience of description, only the portions related to the present application are shown in the drawings. Embodiments of the present disclosure and features of embodiments may be combined with each other without conflict.
It should be noted that the terms "first," "second," and the like in this disclosure are merely used to distinguish between different devices, modules, or units and are not used to define an order or interdependence of functions performed by the devices, modules, or units.
It should be noted that references to "one", "a plurality" and "a plurality" in this disclosure are intended to be illustrative rather than limiting, and those of ordinary skill in the art will appreciate that "one or more" is intended to be understood as "one or more" unless the context clearly indicates otherwise.
The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
With reference to figures 1-4 of the drawings,
a construction method of SPA6 gene comprises the following steps:
designing a carrier and a plurality of mutation primers, and preserving for later use;
the method comprises the steps that a part of mutation primers and a carrier realize that the carrier mutates at a plurality of mutation sites through a PCR technology, a plurality of groups of gene fragments are obtained, and the plurality of groups of gene fragments are recombined to construct a complete gene sequence;
extracting a first gene fragment on the complete gene sequence and a second gene fragment on a vector by using an enzyme digestion technology; and connecting the first gene segment with the second gene segment T4 to construct a plasmid containing a preset gene segment;
performing secondary mutation on the rest mutation primers and the plasmid containing the preset gene fragment by a PCR technology to construct a gene fragment containing the target gene;
obtaining a target gene from the gene fragment containing the target gene by using an enzyme digestion technology, and obtaining a third gene fragment on a plasmid; and connecting the target gene and the third gene fragment through T4 to construct a DomainA-6 containing the target gene;
the sequence of the DomainA-6 gene is shown as SEQ ID NO. 1.
In particular, the method comprises the steps of,
the sequence of the plasmid gene containing the preset gene fragment is shown as SEQ ID NO. 2;
the sequence of the target gene is shown as SEQ ID NO. 3;
the sequence of the third gene segment gene is shown as SEQ ID NO. 4.
In particular, the method comprises the steps of,
the plurality of mutation primers are:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'、DomainAF4'、DomainAR4'。
in particular, the method comprises the steps of,
the partial mutation primer comprises the following components:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'。
in particular, the method comprises the steps of,
the method comprises the steps of carrying out mutation on a plurality of mutation sites by a part of mutation primers and a carrier through a PCR technology, obtaining a plurality of groups of gene fragments, recombining the plurality of groups of gene fragments, and constructing a complete gene sequence, wherein the steps comprise:
carrying out PCR on the DomainAF1', single2-1R, single2-2F, single-2R', single2-3F ', domainAR3' and the vector to realize that the vector is mutated at a plurality of mutation sites and three groups of gene fragments are obtained; the three groups of gene fragments are a first fragment, a second fragment and a third fragment.
Overlapping PCR is carried out on the DomainAF1', the DomainAR3' and the three groups of gene fragments, so that a complete gene sequence is constructed.
Wherein,
carrying out PCR on the DomainAF1', single2-1R, single2-2F, single-2R', single2-3F ', domainAR3' and the vector to realize that the vector mutates at a plurality of mutation sites and obtain three groups of gene fragments, wherein the steps comprise:
PCR was performed on the vector using primers DomainAF1', single2-1R, single2-2F, single2-2R', single2-3F ', domainAR3', respectively;
the PCR system is as follows:
| reagent name | Usage amount |
| PrimeSTAR Max | 20μL |
| F' | 2μL |
| R' | 2μL |
| Plasmid # 6 | 0.5μL |
| H 2 O | 15.5μL |
PCR procedure:
through the above procedure, a first PCR product (SEQ ID No.13,14, 15) was obtained and 10x Loading Buffer was added to the first PCR product for 1% agarose gel electrophoresis, 150V,15min later, the nucleic acid bands were observed and three sets of gene fragments were excised and collected for successful reaction.
Overlapping PCR is carried out on the DovanaF 1', the DovanaR 3' and the three groups of gene fragments to construct a complete gene sequence:
overlapping PCR was performed on the three sets of gene fragments collected
The PCR system is as follows:
PCR procedure:
through the above procedure, a second PCR product (SEQ ID NO. 2) was obtained and 10x Loading Buffer was added to the second PCR product for 1% agarose gel electrophoresis, 150V,15min later, the nucleic acid band was observed and the complete gene sequence was collected by cutting.
Extracting a first gene fragment on the complete gene sequence and a second gene fragment on a vector by using an enzyme digestion technology; and the step of ligating the first gene fragment with the second gene fragment T4 to construct a plasmid containing a predetermined gene fragment comprises:
the vector was double digested with < Xbal, salI >, and digested at 37℃for 2 hours.
The enzyme digestion system is as follows:
simultaneously, double digestion was performed on the complete gene sequence using < Xbal, salI >, at 37℃for 2 hours.
The enzyme digestion system is as follows:
adding 10x Loading Buffer into the mixed product obtained by double enzyme digestion of the carrier to carry out 1% agarose gel electrophoresis, observing nucleic acid strips after 150V and 15min, and cutting gel to collect the carrier successfully subjected to enzyme digestion;
meanwhile, adding 10x Loading Buffer into a mixed product obtained by double enzyme digestion of the complete gene sequence to carry out 1% agarose gel electrophoresis, observing nucleic acid strips after 150V for 15min, and cutting gel to collect the complete gene sequence successfully subjected to enzyme digestion;
t4 ligation was performed on the collected vector and the complete gene sequence.
The connection system is as follows:
t4 connection
pET28a<Xbal,SalI>： 2μL
PCR recovery result < Xbal, salI >: 18 mu L
Solution I: 20μL-40μL
And (3) connecting at 16 ℃ for 5 hours to obtain a connecting product.
DH5 alpha is transformed from the connection product, the mixture is incubated on ice for 30min and then is subjected to heat shock at 42 ℃ for 90s, the mixture is incubated on ice for 2-3min again, 500 mu L of non-antibiotic LB is added and is subjected to shaking culture at 37 ℃ for 40min,4000rpm is used for centrifugation for 5min, 500 mu L of non-antibiotic LB re-suspension bacteria liquid is taken and coated on a Kana plate, and an incubator at 37 ℃ is used for overnight.
The positive monoclonal bacteria P on the Kana plate are selected and screened and subjected to sample feeding sequencing on the next day, and the strain with complete sequencing results is taken to extract plasmids.
The remaining mutation primers and the plasmid containing the predetermined gene fragment are subjected to secondary mutation by PCR technology, so as to construct a gene fragment containing the target gene, which comprises the steps of:
PCR was performed on plasmids using the DomainA F4'/DomainA R4' primers.
The PCR system is as follows:
| reagent name | Usage amount |
| PrimeSTAR Max | 20μL |
| DomainA F4' | 2μL |
| DomainA R4' | 2μL |
| Plasmid # 6 | 0.5μL |
| H 2 O | 15.5μL |
PCR procedure:
through the above process, a third PCR product (SEQ ID NO. 3) was obtained and 10x Loading Buffer was added to the third PCR product to carry out 1% agarose gel electrophoresis, 150V,15min later, the nucleic acid band was observed and the plasmid was cut and recovered to succeed in PCR, thus obtaining the gene fragment containing the target gene.
Obtaining a target gene from the gene fragment containing the target gene by using an enzyme digestion technology, and obtaining a third gene fragment on a plasmid; and the step of constructing the DomainA-6 containing the target gene by connecting the target gene and the third gene fragment through T4 comprises:
the recovered gene fragment containing the target gene was digested with BamHI and SalI, respectively, from the plasmid.
And (3) enzyme cutting system:
adding 10x Loading Buffer into a mixed product obtained by double enzyme digestion of the gene fragment containing the target gene to carry out 1% agarose gel electrophoresis, observing nucleic acid strips after 150V and 15min, and cutting gel to collect the gene fragment containing the target gene which is successfully subjected to enzyme digestion;
meanwhile, adding 10x Loading Buffer into a mixed product obtained by double enzyme digestion of plasmids to carry out 1% agarose gel electrophoresis, observing nucleic acid strips after 150V for 15min, and cutting gel to collect plasmids which are successfully digested;
the plasmid was T4 ligated to a gene fragment containing the target gene.
Transferring the T4 connection product into DH5 alpha, incubating on ice for 30min, then heat-shocking for 90s at 42 ℃, incubating on ice for 2-3min again, adding 500 mu L of non-antibiotic LB, performing shake culture at 37 ℃ for 40min, centrifuging at 4000rpm for 5min, and then coating a Kana plate with 500 mu L of non-antibiotic LB re-suspension, and culturing in a 37 ℃ incubator overnight.
The positive monoclonal bacteria P on the Kana plate are selected and screened and subjected to sample feeding sequencing the next day, and the strain with complete sequencing result is taken to extract plasmid as DomainA-6.
In particular, the method comprises the steps of,
single2-1R and single2-2R' each have bases partially complementary to each other;
both single2-2F and single2-3F' have bases that are partially complementary to each other.
In particular, the method comprises the steps of,
the vector is a single-repeat protein A Domain A SPA 11 protein expression precursor.
In particular, the method comprises the steps of,
the sequence of the DomainAF1' gene is shown as SEQ ID NO. 5;
single2-1R, the sequence of the gene is shown as SEQ ID NO. 6;
single2-2F, the sequence of the gene is shown as SEQ ID NO. 7;
single2-2R' gene sequence is shown in SEQ ID NO. 8;
single2-3F', the sequence of the gene is shown as SEQ ID NO. 9;
the sequence of the DomainAR3' gene is shown as SEQ ID NO. 10;
the sequence of the DomainAF4' gene is shown as SEQ ID NO. 11;
the sequence of the DomainAR4' gene is shown in SEQ ID NO. 12.
In order to verify the alkali resistance of the domainA-6 gene, the domainA-6 gene was expressed as follows:
adding BL21 competent plasmid 1 μL of domainA-6, mixing, incubating on ice for 30min, heat-shocking at 42deg.C for 90s, adding 500mL of antibiotic-free LB, shaking at 37deg.C for 30min, and mixing. LB Kana plates were coated. The positive BL21 strain is inoculated into 3mL of LB culture medium, cultured for 4-5h at 37 ℃, then added with 0.1% IPTG, cultured for 6-12 h at 20 ℃, 1mL of treated and SDS-PAGE running gel (whole bacteria, supernatant and sediment) is taken for detecting the expression condition of the target protein.
After confirming the expression, the expressed protein is transferred to 300mL LB, cultured for 4-5h at 37 ℃, then added with 0.1% IPTG, cultured overnight at 20 ℃, and the expressed mycoprotein is collected.
IPTG is isopropyl beta-D-1-thiogalactoside.
The mycoprotein is treated by 1M NaOH for 24 hours, 8 hours in the middle and 12 hours, and is sampled for 24 hours, and the samples taken out after the treatment are replaced for 3 times and concentrated to 200-400 mu L for activity detection.
The activity test uses a two-way agar diffusion test, agar is dissolved by heating with normal saline to prepare a 1% agar plate, and after perforation, a precipitation line is formed at the corresponding hole according to concentration gradients of 0.5mg/mL,0.25mg/mL,0.125mg/mL,0.0625mg/mL,0.03125mg/mL and 0.0156mg/mL, and the solution is diffused at 37 ℃ for 48 hours.
Activity comparison was performed between the activity of domainA-6 and the activity of rSPA.
Referring to FIG. 4 of the drawings, it can be seen that the domainA-6 prepared according to the present application has much higher protein activity than wild rSPA after a period of time.
The foregoing description is only of the preferred embodiments of the present disclosure and description of the principles of the technology being employed. It will be appreciated by those skilled in the art that the scope of the application in the embodiments of the present disclosure is not limited to the specific combination of the above technical features, but encompasses other technical features formed by any combination of the above technical features or their equivalents without departing from the spirit of the application. Such as the above-described features, are mutually substituted with (but not limited to) the features having similar functions disclosed in the embodiments of the present disclosure.
And (3) a sequence table:
SEQ ID：1：
ATGCACCACCACCATCACCACGTGGACGCGAAATTCGACGCGGATAACAACTTTAACAAAGAGCAGCAGAACGCATTCTATGAAATCCTGAACATGCCGAACCTGAATGAAGAACAGCGTCTGGGCTTCATCCAGTCTCTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCTGAGCGAAGCTAAAAAACTGAACGAAAGCCAGGCGCCGAAGCAGGCGCCGAAAGTTGATGCTAAGTTTGACGCAGACAATAATTTCAATAAGGAACAACAAAATGCGTTTTACGAGATTCTTAATATGCCAAATCTTAACGAGGAGCAACGTCTGGGTTTTATTCAAAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATTATCTGAGGCGAAGAAGTTAAATGAGAGTCAAGCACCTAAACAAGCTCCTAAGGTCGACGCGAAATTCGACGCGGATAACAACTTTAACAAAGAGCAGCAGAACGCATTCTATGAAATCCTGAACATGCCGAACCTGAATGAAGAACAGCGTCTGGGCTTCATCCAGTCTCTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCTGAGCGAAGCTAAAAAACTGAACGAAAGCCAGGCGCCGAAGCAGGCGCCGAAAGTTGATGCTAAGTTTGACGCAGACAATAATTTCAATAAGGAACAACAAAATGCGTTTTACGAGATTCTTAATATGCCAAATCTTAACGAGGAGCAACGTCTGGGTTTTATTCAAAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATTATCTGAGGCGAAGAAGTTAAATGAGAGTCAAGCACCTAAACAAGCTCCTAAGGGATCCGGCGGCGGCTGCTAA。
SEQ ID：2：
TGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGCACCACCACCATCACCACGTGGACGCGAAATTCGACGCGGATAACAACTTTAACAAAGAGCAGCAGAACGCATTCTATGAAATCCTGAACATGCCGAACCTGAATGAAGAACAGCGTCTGGGCTTCATCCAGTCTCTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCTGAGCGAAGCTAAAAAACTGAACGAAAGCCAGGCGCCGAAGCAGGCGCCGAAAGTTGATGCTAAGTTTGACGCAGACAATAATTTCAATAAGGAACAACAAAATGCGTTTTACGAGATTCTTAATATGCCAAATCTTAACGAGGAGCAACGTCTGGGTTTTATTCAAAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATTATCTGAGGCGAAGAAGTTAAATGAGAGTCAAGCACCTAAACAAGCTCCTAAGGTCGACGCGAAA
SEQ ID：3
TCGAGGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCT
SEQ ID：4：
AATGCACCACCACCATCACCACGTGGACGCGAAATTCGACGCGGATAACAACTTTAACAAAGAGCAGCAGAACGCATTCTATGAAATCCTGAACATGCCGAACCTGAATGAAGAACAGCGTCTGGGCTTCATCCAGTCTCTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCTGAGCGAAGCTAAAAAACTGAACGAAAGCCAGGCGCCGAAGCAGGCGCCGAAAGTTGATGCTAAGTTTGACGCAGACAATAATTTCAATAAGGAACAACAAAATGCGTTTTACGAGATTCTTAATATGCCAAATCTTAACGAGGAGCAACGTCTGGGTTTTATTCAAAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATTATCTGAGGCGAAGAAGTTAAATGAGAGTCAAGCACCTAAACAAGCTCCTAAGGTCGACGCGAAAGGATCCGGCGGCGGCTGCTAACTC
SEQ ID：5：
TGAGCGGATAACAATTCCCCTCTAGAA。
SEQ ID：6：
CTGGGACGGATCATCTTTCAGAGACTGGATGAAGCCCAGACGCTGTTCTTCATTCAG。
SEQ ID：7：
CTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCT。
SEQ ID：8：
TTGACTAGGGTCGTCTTTTAGGCTTTGAATAAAACCCAGACGTTGCTCCTCGTTAAG。
SEQ ID：9：
AAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATT。
SEQ ID：10：
CCGCCGCCGGATCCTTTCGCGTCGACCTTAGGAGCTT。
SEQ ID：11：
AATGCACCACCACCATCACCACGTCGACGCGAAATTC。
SEQ ID：12：
GAGTTAGCAGCCGCCGCCGGATCCCTTAGGAGCTTGTTT。
SEQ ID：13：
TGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGCACCACCACCATCACCACGTGGACGCGAAATTCGACGCGGATAACAACTTTAACAAAGAGCAGCAGAACGCATTCTATGAAATCCTGAACATGCCGAACCTGAATGAAGAACAGCGTCTGGGCTTCATCCAGTCTCTGAAAGATGATCCGTCCCAG
SEQ ID：14：
CTGAAAGATGATCCGTCCCAGTCCGCGAACCTGCTGAGCGAAGCTAAAAAACTGAACGAAAGCCAGGCGCCGAAGCAGGCGCCGAAAGTTGATGCTAAGTTTGACGCAGACAATAATTTCAATAAGGAACAACAAAATGCGTTTTACGAGATTCTTAATATGCCAAATCTTAACGAGGAGCAACGTCTGGGTTTTATTCAAAGCCTAAAAGACGACCCTAGTCAA
SEQ ID：15：
AAGCCTAAAAGACGACCCTAGTCAAAGTGCTAATTTATTATCTGAGGCGAAGAAGTTAAATGAGAGTCAAGCACCTAAACAAGCTCCTAAGGTCGACGCGAAAGGATCCGGCGGCGG。
Claims (8)
1. the construction method of the SPA6 gene is characterized by comprising the following steps:
designing a carrier and a plurality of mutation primers, and preserving for later use;
the method comprises the steps that a part of mutation primers and a carrier realize that the carrier mutates at a plurality of mutation sites through a PCR technology, a plurality of groups of gene fragments are obtained, and the plurality of groups of gene fragments are recombined to construct a complete gene sequence;
extracting a first gene fragment (SEQ ID NO. 2) on the complete gene sequence and a second gene fragment on the vector using an enzyme digestion technique; and ligating the first gene fragment with the second gene fragment (SEQ ID NO. 3) T4 to construct a plasmid containing a preset gene fragment;
performing secondary mutation on the rest mutation primers and the plasmid containing the preset gene fragment by a PCR technology to construct a gene fragment containing the target gene;
obtaining a target gene from the gene fragment (SEQ ID NO. 2) containing the target gene by using an enzyme digestion technology, and obtaining a third gene fragment (SEQ ID NO. 4) on a plasmid; and connecting the target gene and the third gene fragment through T4 to construct a DomainA-6 containing the target gene;
the sequence of the DomainA-6 gene is shown as SEQ ID NO. 1.
2. The method for constructing SPA6 gene according to claim 1, wherein:
the sequence of the plasmid gene containing the preset gene fragment is shown as SEQ ID NO. 2;
the sequence of the target gene is shown as SEQ ID NO. 3;
the sequence of the third gene segment gene is shown as SEQ ID NO. 4.
3. The method for constructing SPA6 gene according to claim 1, wherein:
the plurality of mutation primers are:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'、DomainAF4'、DomainAR4'。
4. the method for constructing SPA6 gene according to claim 3, wherein:
the partial mutation primer comprises the following components:
DomainAF1'、single2-1R、single2-2F、single2-2R'、single2-3F'、DomainAR3'。
5. the method for constructing SPA6 gene according to claim 4, wherein:
the method comprises the steps of carrying out mutation on a plurality of mutation sites by a part of mutation primers and a carrier through a PCR technology, obtaining a plurality of groups of gene fragments, recombining the plurality of groups of gene fragments, and constructing a complete gene sequence, wherein the steps comprise:
carrying out PCR on the DomainAF1', single2-1R, single2-2F, single-2R', single2-3F ', domainAR3' and the vector to realize that the vector is mutated at a plurality of mutation sites and three groups of gene fragments are obtained;
overlapping PCR is carried out on the DomainAF1', the DomainAR3' and the three groups of gene fragments, so that a complete gene sequence is constructed.
6. The method for constructing SPA6 gene according to claim 5, wherein:
single2-1R and single2-2R' each have bases partially complementary to each other;
both single2-2F and single2-3F' have bases that are partially complementary to each other.
7. The method for constructing SPA6 gene according to claim 5, wherein:
the vector is a single-repeat protein A Domain A SPA 11 protein expression precursor.
8. The method of constructing SPA6 gene according to any one of claims 3 to 7, wherein:
the sequence of the DomainAF1' gene is shown as SEQ ID NO. 5;
single2-1R, the sequence of the gene is shown as SEQ ID NO. 6;
single2-2F, the sequence of the gene is shown as SEQ ID NO. 7;
single2-2R' gene sequence is shown in SEQ ID NO. 8;
single2-3F', the sequence of the gene is shown as SEQ ID NO. 9;
the sequence of the DomainAR3' gene is shown as SEQ ID NO. 10;
the sequence of the DomainAF4' gene is shown as SEQ ID NO. 11;
the sequence of the DomainAR4' gene is shown in SEQ ID NO. 12.
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| Application Number | Priority Date | Filing Date | Title |
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