Taq DNA polymerase mutant Taq001 and its encoding gene, expression plasmid and prokaryotic expression host

Abstract

The present invention provides a Taq DNA polymerase mutant Taq001, whose sequence is shown in SEQ ID No.3. Compared with the amino acid sequence of the wild-type Taq DNA polymerase shown in SEQ ID NO.1, it has substitutions at the amino acid sites of V14 and G286. Also disclosed are its encoding gene, expression plasmid and prokaryotic expression host. At an extension speed of 10s / kb, the PCR product yield of Taq001 is 5-20 times higher than that of the wild-type Taq DNA polymerase, and the amplification of low-concentration templates can be completed faster; at the same time, compared with the wild-type and truncated (i.e., Klentaq1) Taq DNA polymerases, Taq001 has significant advantages in tolerating immunoglobulins and hemoglobin.

Description

Technical Field

[0001] The patent of this invention relates to a Taq DNA polymerase mutant Taq001 and its encoding gene, expression plasmid, and prokaryotic expression host, and belongs to the field of bioengineering technology. Background Art

[0002] Taq DNA polymerase belongs to the DNA polymerase I family and is currently the most widely used thermostable DNA polymerase. It has become an important tool in scientific research and medical fields. Taq DNA polymerase contains a 5'→3' exonuclease activity domain, which is composed of 1-291 amino acids at the N-terminus. This region also contains binding sites for divalent metal ions. Its C-terminal 424-832 is the 5'→3' polymerase activity domain, which can be divided into the palm region, thumb region and finger region. The palm region is the catalytic center of TaqDNA polymerase, and the thumb region is responsible for binding the primer-template complex, and together with the finger region, it provides support for the stability of the ternary complex and the continuation of the catalytic reaction. In addition, homology analysis results show that the 292-423 amino acids of Taq DNA polymerase contain a sequence similar to the 3'→5' exonuclease activity domain structure, but this region does not have the corresponding activity, resulting in low fidelity of Taq DNA polymerase.

[0003] It is generally believed that the extension speed of wild-type Taq DNA polymerase is about 60s / kb, which is difficult to meet the needs of rapid detection of target genes.

[0004] Although Taq DNA polymerase has long occupied an important position in the field of in vitro diagnosis, its application in special scenarios still has many shortcomings. For example, when there is 0.004% blood by volume in the PCR reaction system, the amplification guided by wild-type Taq DNA polymerase and truncated Klentaq1 DNA polymerase is completely inhibited, and the presence of Ca2+, Mn2+, K+, and Fe3+ will also affect the synthesis of DNA products to varying degrees. At a time when the demand for rapid clinical testing is becoming more and more urgent, blood inhibitors limit the direct application of Taq DNA polymerase. The increased pre-treatment steps not only slow down the detection speed, but may also affect the accuracy and sensitivity of the test.

[0005] The main inhibitors in the blood include immunoglobulins, hemoglobin and hemoglobin. Immunoglobulins can bind to single-stranded DNA, thereby inhibiting primer annealing and preventing the polymerization reaction from starting; hemoglobin and hemoglobin can reversibly block the active site of the polymerase, terminating the polymerization reaction prematurely. Therefore, DNA polymerases that can tolerate the above three main inhibitors will have the hope of completing direct blood amplification and significantly shortening the detection time of pathogens and gene mutations.

[0006] In order to improve the performance of Taq DNA polymerase, some directed evolution methods have been applied to the screening of Taq DNA polymerase, among which the most advantageous is the partitioned self-replication technology. This technology was developed by Ghadessy et al. in 2001. Its main principle is to use the mixture of oil and water to separate the aqueous phase into different small droplets. The aqueous phase contains the components such as dNTP, primers, buffer required for the PCR reaction, while the enzyme and template for amplification are provided by Escherichia coli. By adjusting the input amount of Escherichia coli, it is possible to ensure that there is at most one cell in each droplet. The PCR primers specifically amplify the gene fragment of Taq DNA polymerase to associate the enzymatic activity with the DNA sequence. Usually, a mutant library of Taq DNA polymerase can be pre-constructed by means such as random mutation. Each mutant is expressed in different Escherichia coli and eventually enters different aqueous phase droplets. As long as the appropriate screening pressure is set, the PCR reaction in the droplet can quickly enrich the DNA sequence of the dominant mutant.

[0007] The screening throughput of the compartmentalized self-replication technology can reach up to 10^8 / round, and the types of mutants can be reduced to less than 10 within a few rounds. Since its development, this method has been successfully applied to improve the heat resistance, tolerance of non-natural nucleotides and tolerance of inhibitors of DNA polymerase. Similar technologies with simple modifications can also achieve the evolution of non-DNA polymerases such as RNA polymerase and tRNA synthetase. Summary of the invention

[0008] The purpose of the present invention is to provide a screened Taq DNA polymerase mutant Taq001, which can tolerate blood inhibitors and has an improved extension speed.

[0009] The present invention discloses a Taq DNA polymerase mutant Taq001, the sequence of which is shown in SEQ ID No. 3. Compared with the amino acid sequence of the wild-type Taq DNA polymerase shown in SEQ ID NO. 1, the mutant has substitutions at the amino acid sites of V14 and G286.

[0010] The present invention also discloses the coding gene of the Taq DNA polymerase mutant Taq001, and the sequence thereof is shown as SEQID No.4.

[0011] The invention also discloses an expression plasmid of the Taq DNA polymerase mutant Taq001, and the plasmid vector is pET21b(+).

[0012] The invention also discloses a prokaryotic expression host of the Taq DNA polymerase mutant Taq001, and the expression host is Escherichia coli.

[0013] The beneficial effects of the present invention are as follows: at an extension speed of 10s / kb, the PCR product yield of Taq001 is 5-20 times higher than that of wild-type Taq DNA polymerase, and the amplification of low-concentration templates can be completed faster; at the same time, compared with wild-type and truncated (i.e., Klentaq1) Taq DNA polymerases, Taq001 has significant advantages in tolerance to immunoglobulins and hemoglobin.

[0014] The amino acid sequence of the truncated Taq DNA polymerase is shown in SEQ ID NO.5, and the encoding gene sequence is shown in SEQ ID NO.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The agarose gel electrophoresis diagram of the present invention for testing the tolerance of Taq DNA polymerase to blood inhibitors immunoglobulin and hemoglobin. The immunoglobulin concentration is set to 0 to 50 μg / mL, and the hemoglobin concentration is set to 0 to 2.5 mg / mL. WT is the wild-type Taq DNA polymerase (SEQ ID NO.1), Taq001 is the mutant Taq001 (SEQ ID NO.3), and Klentaq1 is the truncated Taq DNA polymerase (SEQ ID NO.5).

[0016] Figure 2 Agarose gel electrophoresis was used to test the resistance of Taq DNA polymerase to the blood inhibitor porcine hemosiderin for the present invention. The porcine hemosiderin concentration was set at 0.01 to 10 μM.

[0017] Figure 3 The agarose gel electrophoresis for testing the extension speed of Taq DNA polymerase in the present invention, the extension speeds set by the instrument are 10s / kb, 15s / kb, 30s / kb, and 60s / kb, respectively. WT is the wild-type Taq DNA polymerase (SEQ ID NO.1), and 001 is the mutant Taq001 (SEQ ID NO.3).

[0018] Figure 4 Agarose gel electrophoresis was used to test the yield of PCR products using Taq DNA polymerase in the present invention.

[0019] Figure 5 For Figure 4 Grayscale scan quantification of PCR yield. DETAILED DESCRIPTION

[0020] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The embodiments provided are only illustrative of the method of the present invention and do not limit the rest of the content disclosed by the present invention in any way.

[0021] Embodiment 1:

[0022] 1. Using the wild-type Taq DNA polymerase gene sequence as a template (as shown in SEQ ID NO.2), introduce 0.1 mM MnCl into the conventional PCR system. 2 The mutation rate of the PCR reaction process is increased, and a DNA band of about 2500 bp in length is recovered after amplification is completed.

[0023] The primers and reaction conditions for PCR are as follows:

[0024] Taq*-1:5'-GATATACATATGCGCGGCATGCTGCCGCTGTTCG-3'(SEQ ID NO.7)

[0025] Taq*-2:5'-GGTGGTGCTCGAGTTATTCTTTAGCGCTCAGCCA-3'(SEQ ID NO.8)

[0026]

[0027] 2. Using pET21b-Taq vector as template, amplify and recover a DNA product of about 5500 bp. The PCR primers are as follows:

[0028] Ftaq_XXp-F:5'-TGCCGCGCATATGTATATCTCCTTCTTAAAGTT-3'(SEQ ID NO.9)

[0029] Ftaq_XXp-R:5'-GTTGAAGTTGGTATTGGTGAAGATTGGCTGAG-3'(SEQ ID NO.10)

[0030] 3. Perform DNA in vitro recombination reaction at 50°C with a molar ratio of exogenous fragment to vector = 3:1, and the total reaction volume is 400 μL. After 1 hour, the recombinant product is purified by column according to the standard process and finally eluted with 20 μL ddH2O.

[0031] 4. Electrotransform the eluted product into competent E. coli BL21 (DE3) cells, and quickly add 900 μL of pre-cooled fresh LB medium after 3.0 kV, 60 ms electric pulse. Mix by blowing and pipetting all the liquid into the EP tube, and place it in an ice water bath for more than 2 minutes. Transfer the EP tube to a 37°C shaker, and resuscitate at 250 rpm for 1 hour. Take 10 μL of the resuscitated bacterial solution, dilute it, and spread it all on the solid LB medium containing carbenicillin. The remaining bacterial solution is inoculated into 20 mL of liquid LB medium containing carbenicillin.

[0032] 5. According to the plate counting results, the capacity of the Taq mutant library was estimated to be about 5*10^6. The sequencing results showed that there were about 2*10^6 effective mutants and the mutation frequency was 1-5 / kb.

[0033] 6. Inoculate the library into 20 mL of liquid LB medium at a ratio of 2%, culture at 37°C and 250 rpm until the OD600 value is about 0.8, then add 0.5 mM IPTG to induce protein expression. Collect the cells after 4 hours, centrifuge the cell solution containing 1 OD of cells, and wash twice with 1× reaction buffer.

[0034] 7. Prepare the reaction solution and water-in-oil droplets according to the standard procedure, and then dispense the droplets into 200 μL eight-tube tubes at 50 μL / tube. Perform the PCR reaction in the droplets according to the following conditions, and purify the DNA products in the droplets after the reaction. Perform recovery PCR using the purified DNA as a template to obtain a sufficient amount of DNA product. The reaction system and conditions are as follows:

[0035] a. Water phase system in droplets:

[0036] Components volume D21pcr_fwd / D21pcr_rev 6.0μL / 6.0μL 10*Reaction Buffer 30μL dNTP Mix (2.5 mM each) 48μL Inhibitor 20μL E. coli 100μL ddH2O to 300μL

[0037] Among them, D21pcr_fwd:5'-CAGGAAACAGCTATGACAAAATCGAGCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTT-3'(SEQ ID NO.11)

[0038] D21pcr_rev:5'-AAACGACGGCCAGTACATCAGCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTG-3'(SEQ ID NO.12)

[0039] b. In-droplet PCR conditions:

[0040]

[0041] c. Recovery PCR conditions:

[0042]

[0043] Recover PCR primers as follows:

[0044] Adapter_Fwd:5'-CAGGAAACAGCTATGACAAAATCGAG-3'(SEQ ID NO.13)

[0045] Adapter_rev:5'-AAACGACGGCCAGTACATCAGC-3'(SEQ ID NO.14)

[0046] 8. The recovered PCR products were electrophoresed, and the DNA fragments of about 2800 bp were collected by column purification. The DNA fragments were digested with XhoI / XbaI and connected with pET21b(+) vector, and then transformed into BL21(DE3) competent cells again to enter the next round of compartmentalized self-replication cycle. The screening conditions for each cycle are as follows:

[0047] Number of cycles Inhibitor working concentration (μg / mL) Amplification speed (s / kb) 1 0 60 2 5 60 3 5 45 4 10 30 5 20 30 6 20 20 7 40 20 8 40 10 9 50 10

[0048] 9. Determine the gene sequences of several single clones in each cycle, analyze the sequence enrichment, and finally obtain the preferred mutant sequence, one of which is shown in SEQ ID NO.4.

[0049] 10. The Escherichia coli containing the sequences of SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 were cultured and induced to express Taq DNA polymerase. The cells were collected, washed, resuspended, and then subjected to ultrasonic and heat treatment to break the cell wall to release the Taq DNA polymerase into the solution. Finally, high-purity Taq DNA polymerase was obtained by purification through a heparin column.

[0050] Embodiment 2:

[0051] Prepare immunoglobulin solutions of different concentrations for later use, and configure the PCR reaction according to the following system, adding 3U of purified different Taq DNA polymerases to each 50μL reaction:

[0052] Components volume D21pcr_fwd / D21pcr_rev 1.0μL / 1.0μL 10*Reaction Buffer 5μL dNTP Mix (2.5 mM each) 8μL Inhibitor 10μL Taq DNA polymerase 3U <![CDATA[ddH 2 The]]> to 50μL

[0053] like Figure 1 As shown on the left, different Taq DNA polymerases can all tolerate 10 μg / mL of human immunoglobulin, but only Taq001 can tolerate 50 μg / mL of inhibitors to a certain extent. That is, after targeted screening of the separated self-replication system, the inhibitor tolerance of Taq DNA polymerase has been improved.

[0054] The other two major inhibitors in the blood are hemoglobin and hemoglobin, and the resistance of Taq DNA polymerase to them is shown in Figure 1 Right picture and Figure 2 As shown. The results show that the truncated Klentaq1 has the worst tolerance to inhibitors. For hemoglobin, Taq001 has a slight yield advantage over the wild type under the condition of 2.5 mg / mL. However, Taq001 is less tolerant to porcine hemoglobin than the wild type Taq DNA polymerase.

[0055] The PCR conditions used to test tolerance were as follows:

[0056]

[0057] Embodiment 3:

[0058] The purified Taq DNA polymerase was taken to test its extension speed. After the PCR system was prepared, the extension time was adjusted to examine the difference in extension speed of different Taq DNA polymerases. Figure 3 As shown in the figure, both wild-type and Taq001 DNA polymerases can complete product amplification at speeds of 60s / kb, 30s / kb, and 15s / kb, and the wild-type Taq has a higher yield than Taq001. Under the condition of 10s / kb, the amplification product yield of Taq001 is significantly higher than that of wild-type Taq DNA polymerase, indicating that the amplification speed of Taq DNA polymerase has been improved to a certain extent.

[0059] In order to test the specific performance differences of the two enzymes under the 10s / kb condition, different units of enzyme protein were added to the 50μL system for PCR reaction. Figure 4 As shown in the figure, the amplification products of both enzymes increased with the increase of protein concentration, and the product yield of Taq001 was higher than that of wild-type Taq DNA polymerase. With the help of grayscale scanning quantitative software, the DNA bands under different conditions were relatively quantitatively analyzed. Figure 5 As shown, the yield of Taq001 is 5.38, 8.76, and 19.6 times that of wild-type Taq, respectively. That is, under the same conditions, the Taq DNA polymerase mutant of the present invention can obtain more DNA products and achieve faster detection of the target product.

Claims

1. A Taq DNA polymerase mutant Taq001, characterized in that: Its amino acid sequence is shown in SEQ ID No.

3.

2. The coding gene of the Taq DNA polymerase mutant Taq001 according to claim 1, whose nucleotide sequence is shown in SEQ ID No.

4.

3. An expression plasmid containing the gene encoding the Taq DNA polymerase mutant Taq001 according to claim 2.

4. The expression plasmid according to claim 3, characterized in that The plasmid vector is pET21b(+).

5. A prokaryotic expression host containing the gene encoding the Taq DNA polymerase mutant Taq001 according to claim 2, characterized in that The expression host is Escherichia coli.

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