pET28a-NS3-ZIKA Virus

Composite Part: BBa_K4862005 (pET-NS3-ZIKA)

Construction Design

We designed enzyme digestion to connect the ZIKA gene (BBa_K4862001) with the PET28a (BBa_K3521004) plasmid. To do that, we first obtained the NS3-ZIKA through PCR cloning. Then, we used double enzyme digestion with BamHI, XhoI, CutSmart, and ddH2O to connect the ZIKA gene with PET28a.

Engineering Principle

Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is neither a vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation [1].

RNA helicase is responsible for almost all metabolism of RNA; in the Dengue virus, NS3 protein contains the activity of multiple enzymes, including helicase. Inserting the NS3 sequence is for expressing the protein we need and testing whether the medicine finally injected would be effective. The other structure of the pET28a plasmid would not be changed.

While these two plasmids are constructed, they would be transplanted into BL21 bacteria, which can express the NS3 protein we need. After growing for a few hours, the solution (these two plasmids and the liquid medium) would be ultrasonically crushed, destroying the bacteria's basic structure. The answer, consisting of the proteins and bacteria fragments, would be purified for extracting the NS3 proteins.

The NS3 proteins have the ability as a Helicase, which would activate the helicase to work. The protein would be added to a fluorescence DNA vector to observe whether the helicase works. If so, the fluorescence would disappear; if not, the fluorescence would be observed, which also means the medicine for restraining the replication of DNA is working. As NS3 proteins are the main protein active helicase in the Dengue virus, if the ability is restricted, the duplication of the RNA of the bacteria would be stopped, which means the spread of the virus would be halted.

Experimental Approach

Per the construction design, we obtained the plasmid pET-NS3-ZIKA. To express the DNA as protein, we used heat shock conversion that inserts PET28a-NS3-ZIKA into BL21(DE3), a type of Escherichia coli. After this, we transplanted the bacteria by touching the single colony linings of PET-NS3-ZIKA with the pipette gun and leaving the pipette in the LB broth for the 16-hour incubation.

Then, we amplified our target ZIKA DNA sequence by PCR and extracted the plasmid with buffer SP1, SP2, SP3, washing solution, and water. Electrophoresis gel was conducted to validate the successful result, the strip sizes of PET-NS3-ZIKA were detected in 1854 bp. In addition, the sequencing result also supported this conclusion.

Figure 2. Electrophoresis gel and sequencing of pET-NS3-ZIKA

Since we have obtained the BL21 pET-NS3-ZIKA, firstly we need to check if this part pET-NS3-ZIKA could work as expected to express the protein. We used IPTG to express the ZIKA gene through BL21(DE3) while preparing the 15% SDS-PAGE gel. We broke the bacteria with an ultrasonic cell breaker device. We collected the unpurified ZIKA protein solution by adding HIs buffer A to the centrifuge. To purify the protein solution, we utilized Ni-NTA His Tag Purification. Per the SDS-PAGE shown in Figure 4, the protein NS3-ZKA was detected at around 49 kDa.

Figure 3. SDS-PAGE gel indicating NS3-ZIKA’s kDa value

Characterization/Measurement

Secondly, we further used the FRET method to check if the protein NS3-ZIKA possesses helicase activity. We added fluorescence and ATP into the NS3-ZIKA pure protein solution to test whether the helicase is inactive. According to the pET-28a-NS3-ZIKA curve compared to the control pET-28a, the fluorescence value decreases, whereas protein concentration increases. Therefore, we can prove that the NS3 protein possesses helicase activity.

Figure 4. FRET curves of pET-28a-NS3-ZIKA and pET-28a

*Our team measured the fluorescence using fluorometers with an excitation of 620 nm and an emission wavelength of 685 nm.

References

Andrade MA, Mottin M, Sousa BKP, Barbosa JARG, Dos Santos Azevedo C, Lasse Silva C, Gonçalves de Andrade M, Motta FN, Maulay-Bailly C, Amand S, Santana JM, Horta Andrade C, Grellier P, Bastos IMD. Identification of novel Zika virus NS3 protease inhibitors with different inhibition modes by integrative experimental and computational approaches. Biochimie. 2023 Sep;212:143-152. doi: 10.1016/j.biochi.2023.04.004. Epub 2023 Apr 22. PMID: 37088408.

Sequence and Features

Assembly Compatibility: