A kind of chimeric capping enzyme and its preparation method and application
A chimera and seed technology, applied in the field of bioengineering, can solve the problems of low expression and purification efficiency, high price, and restrictions on industrial production, and achieve excellent thermal stability, high protein expression, and excellent enzyme activity.
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[0051] The present invention also provides a method for preparing the chimeric capping enzyme, which specifically includes the following steps:
[0052] (1) Prepare the recombinant expression vector of the chimeric capping enzyme.
[0053](2) Take the recombinant expression vector of the chimeric capping enzyme, inoculate it in 2YT medium (containing 50 μg / mL kanamycin sulfate), and shake to culture to obtain a seed solution. The seed solution was inoculated into a new 2YT medium (containing 50 μg / mL kanamycin sulfate) at a volume ratio of 1:100, and the shaking culture was carried out again to OD. 600 =0.6~0.8, get bacterial liquid. The conditions for shaking culture are preferably: 37°C, 150-200 r / min.
[0054] (3) IPTG was added to the bacterial solution to a final concentration of 0.1-0.5 mM, preferably the final concentration of IPTG was 0.1 mM, induced at 16°C for 18 hours to induce cells to express proteins, and then centrifuged at 4000g at 4°C After 30 min, the cell...
Embodiment 1
[0059] Example 1: Preparation and purification of various chimeric capping enzymes
[0060] 1. Using conventional technical means in the art, construct a variety of chimeric capping enzyme expression vectors, specifically including: VACE (chimeric capping enzyme of vaccinia virus and African swine fever virus), and its protein sequence is as shown in SEQ ID NO.1. VFCE (chimeric capping enzyme of vaccinia virus and amoeba virus), its protein sequence is shown in SEQ ID NO.2; VMCE (chimeric capping enzyme of vaccinia virus and giant virus), its protein sequence is shown in SEQ ID NO. shown in SEQ ID NO.3. At the same time, construct the expression vectors of unchimeric capping enzymes, including: vaccinia virus capping enzyme (VCE), African swine fever virus capping enzyme (ACE), amoeba virus capping enzyme (FCE) and giant virus Capped Enzyme (MCE).
[0061] 2. Transform each capping enzyme and chimeric capping enzyme recombinant expression vector of step 1 into the host cell ...
Embodiment 2
[0067] Example 2: Comparative display of enzymatic activity and capping rate of each capping enzyme
[0068] 1. TPase activity detection. in 50 μL of 1x RNA capping buffer (50 mM Tris HCl, 5 mM KCl, 1 mM MgCl 2 , 1mM DTT, pH=8.0), 1 μg of the capping enzyme or the chimeric capping enzyme of step 1 of Example 1 was incubated with 0.5 mM GTP for 60 minutes at 37°C, respectively. TPase reactivity was analyzed using high performance liquid chromatography (HPLC). The peak areas corresponding to GTP and GDP, respectively, were quantified and used to calculate the relative activity of TPase at the end of the 60-minute capping reaction.
[0069] 2. MTase activity detection. in 20 μL of 1x RNA capping buffer (50 mM Tris HCl, 5 mM KCl, 1 mM MgCl 2 , 1mM DTT, pH=8.0) was additionally added with 2mM SAM, and 1 μg of the capping enzyme or chimeric capping enzyme (7 kinds in total) of step 1 of Example 1 was incubated with 0.5mM GpppA for 60 minutes at 37°C. MTase reactivity was analyz...
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