CN114317485B

CN114317485B - Recombinant murine leukemia virus reverse transcriptase mutant, preparation method and application

Info

Publication number: CN114317485B
Application number: CN202111651558.2A
Authority: CN
Inventors: 刁含文
Original assignee: Nanjing Jujiang Biotechnology Co ltd
Current assignee: Nanjing Jujiang Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-04-11
Anticipated expiration: 2041-12-30
Also published as: CN114317485A

Abstract

The invention relates to the technical field of biology, in particular to a recombinant murine leukemia virus reverse transcriptase mutant, a preparation method and application thereof. The recombinant murine leukemia virus reverse transcriptase mutant provided by the invention improves the thermal stability of the wild type recombinant murine leukemia virus reverse transcriptase by carrying out point mutation molecular modification on the enzyme, and simultaneously improves the reverse transcription efficiency of the product and the reverse transcription capability of long-fragment cDNA. In addition, the invention also provides a recombinant expression vector capable of expressing the mutant, and the recombinant expression vector can efficiently express the mutant after being transformed into escherichia coli.

Description

Recombinant murine leukemia virus reverse transcriptase mutant, preparation method and application

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant murine leukemia virus reverse transcriptase mutant, a preparation method and application thereof.

Background

Reverse transcriptase (Reverse transcriptase) is an enzyme that directs deoxynucleotide triphosphate synthesis of complementary DNA (cDNA) using RNA as a template. Reverse transcriptase (M-MLV) was isolated from Moloney Murine Leukemia Virus and has been genetically altered to remove its associated ribonuclease H activity (an enzyme that specifically cleaves RNA molecules on hybridized strands of DNA and RNA) and used for first strand synthesis of cDNA and primer extension. This enzyme requires magnesium ions or manganese ions as cofactors, and when mRNA is used as a template, single-stranded DNA (ssDNA) is synthesized first, then under the action of reverse transcriptase and DNA polymerase I, "hairpin" -type double-stranded DNA (dsDNA) is synthesized using the single-stranded DNA as a template, and then cleaved into two single-stranded double-stranded DNAs with nuclease S1.

Thus, reverse transcriptase can be used to reverse transcribe the mRNA of any gene into a cDNA copy, and the cDNA inserted into the vector can then be amplified in large quantities. The cDNA can also be labeled as a radioactive molecular probe. M-MLV reverse transcriptase is currently used for first strand cDNA synthesis, cDNA probe generation, RNA transcription, sequencing and RNA reverse transcription reactions. The enzyme deletes RNaseH activity by point mutation, has the same activity as wild type DNA polymerase and obviously improves the extending capability.

However, the current wild-type reverse transcriptase lacking RNaseH activity still has certain defects, mainly manifested by poor reverse transcription capability for large-fragment cDNA and poor thermostability.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a novel recombinant murine leukemia virus reverse transcriptase mutant, so that the mutant can solve the technical problems of poor reverse transcription capability and thermal stability of reverse transcriptase M-MLV on large-fragment cDNA in the prior art.

It is another object of the present invention to provide a biomaterial encoding the above mutant and a method for preparing the mutant using the biomaterial, which enables mass production of the mutant product having purity and activity satisfying market requirements.

The present invention also aims at applying the obtained mutant product in reverse PCR of new coronavirus SARS-CoV-2 or preparing product for reverse PCR of new coronavirus SARS-CoV-2 to provide sufficient technological support for medicine development, medicine screening and scientific research of new coronavirus SARS-CoV-2.

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

in a first aspect, the present invention provides a recombinant murine leukemia virus reverse transcriptase mutant, said mutant being obtained by the replacement of at least one amino acid residue with a wild-type recombinant murine leukemia virus reverse transcriptase having the amino acid sequence as depicted in SEQ ID No. 1.

In an alternative embodiment, the mutant has the amino acid sequence as set forth in SEQ ID No. 2.

In a second aspect, the present invention provides a biomaterial comprising any one of:

a nucleic acid molecule encoding a mutant according to any one of the preceding embodiments;

(ii) a recombinant vector comprising (i) said nucleic acid molecule, said recombinant vector having the original vector pET22b;

(iii) a transformant comprising (i) said nucleic acid molecule or (ii) said recombinant vector, said transformant host being selected from the group consisting of E.coli.

In alternative embodiments, the e.coli is selected from e.coli Rosetta or e.coli BL21.

In a third aspect, the present invention provides a method for preparing a mutant according to any one of the preceding embodiments, comprising constructing a recombinant expression vector of wild-type recombinant murine leukemia virus reverse transcriptase, amplifying to obtain a linear recombinant expression vector DNA, subjecting the linear recombinant expression vector DNA to site-specific mutagenesis, cyclizing with a homologous recombinase to obtain a recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase, transforming the recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase into host bacteria, fermenting and culturing, disrupting the bacterial cells, and collecting the mutant recombinant murine leukemia virus reverse transcriptase.

In an alternative embodiment, two site-directed mutagenesis is performed using two sets of primer pairs, wherein the nucleotide sequence of the forward primer of the first primer pair is set forth in SEQ ID No.3 and the nucleotide sequence of the reverse primer is set forth in SEQ ID No. 4; the nucleotide sequence of the forward primer of the second primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 6.

In an alternative embodiment, the nucleotide sequence of the forward primer used for said amplification is shown as SEQ ID No.7 and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 8.

In an alternative embodiment, the original vector of the recombinant expression vector is pET22b.

In an alternative embodiment, the cognate recombinase is UvsXase and the host bacterium is selected from e.coli Rosetta or e.coli BL21.

In a fourth aspect, the present invention provides the use of a mutant according to any of the preceding embodiments in the qPCR of SARS-CoV-2 virus or in the preparation of a product of the qPCR of SARS-CoV-2 virus.

In an alternative embodiment, the SARS-CoV-2 virus qPCR product comprises a positive primer having a nucleotide sequence as shown in SEQ ID No.9 and a negative primer having a nucleotide sequence as shown in SEQ ID No. 10.

The recombinant murine leukemia virus reverse transcriptase mutant provided by the invention realizes the improvement of the thermal stability of the mutant by carrying out point mutation molecular modification on the wild type recombinant murine leukemia virus reverse transcriptase, and also improves the reverse transcription efficiency of the product and the reverse transcription capability of long-fragment cDNA. In addition, the invention also provides a recombinant expression vector capable of expressing the mutant, and the recombinant expression vector can efficiently express the mutant after being transformed into escherichia coli.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram showing mutation sites of mutant M-MLV provided in example 1 of the present invention;

FIG. 2 shows the result of PCR experiment for M-MLV point mutation in example 1 of the present invention;

FIG. 3 is SDS-PAGE results of mutant M-MLVs expressed in examples 2 and 3 of the present invention;

FIG. 4 shows the reverse transcription cDNA yields under different temperature conditions in Experimental example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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 some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. 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.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In a particular embodiment, in a first aspect, the present invention provides a recombinant murine leukemia virus reverse transcriptase mutant, said mutant being obtained by substitution of at least one amino acid residue of a wild-type recombinant murine leukemia virus reverse transcriptase having the amino acid sequence as depicted in SEQ ID No. 1.

It is to be understood that the wild-type recombinant murine leukemia virus reverse transcriptase of the present invention includes naturally expressed reverse transcriptase of recombinant murine leukemia virus, and may also include reverse transcriptase obtained by modifying the prior art for other defects of the wild-type recombinant murine leukemia virus reverse transcriptase different from the modification of the object of the present invention, for example, amino acid sequence obtained by simple mutation in the prior art for losing RNase activity of the wild-type recombinant murine leukemia virus reverse transcriptase, may be used as the modified material of the present invention because it does not affect the thermostability and reverse transcription ability of the long fragment of the wild-type recombinant murine leukemia virus reverse transcriptase, and should be considered to be included in the wild-type recombinant murine leukemia virus reverse transcriptase of the present invention. The above-described SEQ ID No.1 of the present invention should be understood as an example only, and therefore, the open-ended writing manner adopted should be acceptable.

Meanwhile, the substitution of at least one amino acid residue in the present invention refers to the substitution of amino acid residues for the purpose of improving the thermal stability and reverse transcription ability of the recombinant murine leukemia virus reverse transcriptase, and any amino acid point mutation that can achieve the above functions is within the scope of the present invention.

It should be understood that the number of point mutations that can improve the thermostability and the reverse transcription ability of the recombinant murine leukemia virus reverse transcriptase is enormous and not exhaustive, and as an example, the present invention provides a combination of point mutations of G55A and F189L shown in the amino acid sequence shown in SEQ ID No. 2.

(ii) a recombinant vector comprising the nucleic acid molecule of (i), wherein the original vector of the recombinant vector is pET22b;

It is understood that the sequence of the above-mentioned nucleic acid molecule is not limited to one due to the correspondence between the amino acid residues of the base codon, and any nucleic acid molecule capable of encoding the above-mentioned mutant should be understood as the scope of protection of the above-mentioned nucleic acid molecule.

In order to obtain the mutant, the three-dimensional structure of the obtained wild type recombinant M-MLV is analyzed through a homologous model, and then the site-directed mutation is carried out on the recombined wild type M-MLV through a point mutation technology. Then, a proper vector is selected to construct to obtain a recombinant vector mutant, and finally, the correctly mutated recombinant expression vector is transformed into escherichia coli for protein expression.

In a third aspect, the present invention provides a method for preparing a mutant according to any one of the preceding embodiments, comprising constructing a recombinant expression vector of wild-type recombinant murine leukemia virus reverse transcriptase, amplifying the recombinant expression vector to obtain a linear recombinant expression vector DNA, subjecting the linear recombinant expression vector DNA to site-specific mutagenesis, cyclizing the site-specific mutagenesis with a homologous recombinase to obtain a recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase, transforming the recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase into host bacteria, fermenting and culturing the host bacteria, disrupting the bacteria, and collecting the mutant recombinant murine leukemia virus reverse transcriptase.

In an alternative embodiment, the homologous recombinase is UvsXase and the host bacterium is selected from e.coli Rosetta or e.coli BL21.

In a fourth aspect, the present invention provides the use of a mutant according to any one of the preceding embodiments in a SARS-CoV-2 virus qPCR, or in the preparation of a SARS-CoV-2 virus qPCR product.

Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments and features of the embodiments described below can be combined with each other without conflict.

Example 1

The three-dimensional structure of wild type recombinant M-MLV is simulated, the amino acid sequence of the three-dimensional structure is shown as SEQID No.1, and two mutation points are determined to be G55A and F189L. And the recombinant expression vector formed by the mutated recombinant M-MLV mutant and the vector pET22b is transformed into escherichia coli, and then positive escherichia coli is obtained by screening and used for expressing the recombinant M-MLV mutant, and the specific steps are as follows:

(1) The amino acid sequence of the wild type recombinant M-MLV is modeled by Swiss-Model software to obtain a three-dimensional structure, and two mutation target sites of G55A and F189L are determined, wherein the point mutation sites are shown in figure 1.

(2) Cloning the recombinant M-MLV nucleic acid sequence by using a site-directed Mutagenesis Kit (CloneUFO Fast Mutagenesis Kit) of Nanjing Jumbo Biotech Co., ltd, amplifying DNA of a wild-type recombinant M-MLV expression vector pET-22b (+)/M-MLV according to the instruction to obtain a linear form recombinant expression vector DNA, and cyclizing by using a homologous recombinase UvsXase (Nanjing Jumbo Biotech Co., ltd) to obtain a complete mutant reverse transcriptase recombinant expression vector. Samples were made using the following set of site-directed mutagenesis primer sets (1) to mutate Gly at position 55 to Ala (SEQ ID No.3 and SEQ ID No. 4), and sample (2) to mutate Gly at position 55 to Ala (SEQ ID No.3 and SEQ ID No. 4) while mutating Phe at position 189 to Leu (SEQ ID No.5 and SEQ ID No. 6). The PCR cloning conditions were: 94 ℃ 5min,95 ℃ 15s,58 ℃ 15s,72 ℃ 2min,72 ℃ 10min,10 ℃ infinity. The gel electrophoresis result of the PCR product is shown in FIG. 2, and it is seen from FIG. 2 that the size of the bands obtained by gel electrophoresis of the PCR product is equal before and after the point mutation of G55A alone in the sample (1) and the point mutation of G55A and F189L simultaneously in the sample (2), which proves that the size of the mutant linear vector obtained by PCR is correct.

TABLE 1 primer pairs for point mutations in example 1

Example 2

In this example, the recombinant expression vector of mutant reverse transcriptase obtained in example 1 was transformed into E.coli Rosetta to obtain transformed strain Mut-1/E.coli Rosetta, which was inoculated into 20mL LB liquid medium containing 100mg/mL ampicillin and shake-cultured at 37 ℃ and 220rpm for 12 hours to obtain a seed solution. Inoculating 100 μ L seed solution into 100mL LB liquid medium containing 100mg/mL ampicillin, shake culturing at 37 deg.C and 220rpm until the fermentation broth bacterial concentration reaches OD600=0.6, adding IPTG to 100mg/mL, and inducing expression at 16 deg.C and 180rpm for 16h.

Then, the cells collected by the inducible expression were resuspended in a phosphate buffer (50 mmol/L PBS +0.3mol/L NaCl +0.5% Triton X-100), disrupted by a cell homogenizer (700bar 5 min), centrifuged at 9000rpm for 10min at 4 ℃ and the supernatant was collected as a crude enzyme solution. And (3) eluting the crude enzyme solution obtained by treatment with an eluent containing imidazole with different concentrations (50 mmol/L, 100mmol/L, 150mmol/L and 200 mmol/L) in sequence according to the specification of the Ni-NTA His Tag Kit, and collecting elution peaks to obtain the mutant recombinant murine leukemia virus reverse transcriptase.

Example 3

This example also provides a method for preparing a mutant recombinant murine leukemia virus reverse transcriptase, which differs from example 2 only in that e.coli Rosetta is replaced with e.coli BL21.

The result of the mutant recombinant murine leukemia virus reverse transcriptase expressed by E.coli Rosetta or E.coli BL21 provided by the invention is shown in FIG. 3, and the expression level of E.coli Rosetta is higher than that of E.coli BL21.

Experimental example 1

This example examined the effect of temperature on the cDNA-obtaining effect of the mutant recombinant murine leukemia virus reverse transcriptase and the wild-type recombinant murine leukemia virus reverse transcriptase obtained in example 2, reverse transcribing the SARS-CoV-2RNA, a novel coronavirus.

(1) The extraction method of RNA comprises the following steps: ATGPure of Biotechnology Ltd of Nanjing Judge ^TM RNA was extracted from a commercially available sample of a novel coronavirus by using the Virus RNA Extraction Kit (Column) (RV 301) Kit method.

(2) After the obtained RNA was passed through the purity and concentration test, cDNA was obtained by reverse transcription using the mutant recombinant murine leukemia virus reverse transcriptase obtained in example 2 and the wild-type recombinant murine leukemia virus reverse transcriptase under the following conditions:

preparing the following mixed solution in an RNase-free centrifuge tube:

* The experiment needs to add RNase inhibitor, and ATG (enzyme-linked immunosorbent assay) from Nanjing Judge Biotech Co ^TM Rnasin(ATG#RC102)。

The procedure was as follows:

then, 10. Mu.l of the obtained cDNA sample was subjected to 1% agarose nucleic acid electrophoresis together with a cDNA sample obtained using wild-type M-MLV (amino acid sequence shown in SEQ ID No. 1). The cDNA yields obtained by reverse transcription are shown in FIG. 4, which shows that the cDNA synthesis yields of the mutants are higher than those of the wild type at different reaction temperatures and that they are efficiently amplified at 65 ℃.

Experimental example 2

In this experimental example, the effect of cDNA obtained by reverse transcription of the mutant recombinant murine leukemia virus reverse transcriptase and the wild-type recombinant murine leukemia virus reverse transcriptase obtained in example 2 for fluorescent quantitative qPCR reaction was examined, and the detection was performed by using commercially available N gene of a novel coronavirus as a detection target according to the following steps:

reverse transcription at different temperatures to obtain cDNA template, and using N gene primers N-F and N-R of new coronavirus according to ATGStart of Biotech Co., ltd of Kyoto ^TM Plus qPCR SYBR Green Master Mix(Q111)Methods qPCR experiments were performed.

New crown N gene primer:

N-F：CAGTAGGGGAACTTCTCCTGCTAGAATGGCTGG(SEQ ID No.7)；

N-R：CCTTTACCAGACATTTTGCTCTCAAGCTGG(SEQ ID No.8)；

the reaction Buffer was configured as follows:

qPCR experimental procedure was as follows:

the qPCR amplification results were as follows:

the qPCR result shows that the mutant has lower Ct value and higher detection sensitivity at the same temperature compared with the wild type, and the template can still be detected at the high temperature of 65 ℃, which indicates that the mutant reverse transcriptase has better heat resistance.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

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Claims

1. A recombinant murine leukemia virus reverse transcriptase mutant, characterized in that the amino acid sequence of the mutant is shown in SEQ ID No. 2.

2. Biomaterial, characterized in that it comprises any of the following:

a nucleic acid molecule encoding the mutant of claim 1;

(iii) a transformant comprising (i) said nucleic acid molecule or (ii) said recombinant vector, said transformant host being selected from the group consisting of Escherichia coli (E. Coli)Escherichia coli）。

3. The biomaterial of claim 2, wherein the E.coli is selected fromEscherichia coliRosetta orEscherichia coli BL21。

4. The method of preparing the mutant according to claim 1, wherein the method comprises constructing a recombinant expression vector of wild-type recombinant murine leukemia virus reverse transcriptase, amplifying to obtain a linear recombinant expression vector DNA, subjecting the linear recombinant expression vector DNA to site-specific mutagenesis, cyclizing with a homologous recombinase to obtain a recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase, transforming the recombinant expression vector of mutant recombinant murine leukemia virus reverse transcriptase into host bacteria, fermenting and culturing, disrupting the bacterial cells, and collecting the mutant recombinant murine leukemia virus reverse transcriptase.

5. The method according to claim 4, wherein two sets of primer pairs are used for the two site-directed mutagenesis, wherein the nucleotide sequence of the forward primer of the first primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 4; the nucleotide sequence of the forward primer of the second primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 6.

6. The method of claim 5, wherein the original vector of the recombinant expression vector is pET22b.

7. The method according to claim 6, wherein the homologous recombinase is UvsXase and the host bacterium is selected from the group consisting ofEscherichia coliRosetta orEscherichia coli BL21。

8. Use of the mutant of claim 1 in the preparation of a SARS-CoV-2 virus qPCR product.

9. The use according to claim 8, wherein the SARS-CoV-2 virus qPCR product comprises a positive primer having a nucleotide sequence shown as SEQ ID No.7 and a negative primer having a nucleotide sequence shown as SEQ ID No. 8.