Modify the virus

To facilitate the identification and screening of cells containing the mutated virus in subsequent experiments, we plan to insert GFP along with two forward and reverse promoters (which specifically function in vaccinia virus) between the A27L gene and the A28L gene fragment of the viral genome.

Based on the ideas and templates provided by the Wuhan Institute of Virology (specific to vaccinia virus), we identified the gene sequence of A27L, introduced mutations after the forward codon, placed the GFP gene fragment after the reverse codon, and then added 500bp homologous arms at both ends of the A27L gene fragment (including the A26L gene) and the GFP gene fragment (including the A28L gene).

Under the guidance of our instructor, we modified the A27L gene fragment (as the initially used A27L gene fragment strain differed from the laboratory strain), changed the type of GFP, and adjusted the position of the homologous arms from the end of A27L to the last mutation site on A27L. We then had Qingke Biotechnology Company synthesize this DNA fragment and inserted it into pcDNA3.1-6X Myc.

After discussion, we determined that performing homologous recombination to replace the A27L gene fragment would be a better approach. We then identified the A27L gene sequence of the strain we were using and replaced the three base sequences corresponding to phenylalanine at the N-terminus with ATG. Subsequently, we constructed a plasmid that ensures GFP expression only when infected with vaccinia virus. The plasmid includes basic components, the GFP gene, the mutated A27L gene, vaccinia virus-specific promoters, and 500bp homologous arms required for homologous recombination at both ends.

1. The construction of the plasmid pcDNA3.1-A27Lmut-GFP

2. The virus cloning

Preliminary determination indicates that after the eighth round of infection, the concentration of the modified VTT is higher and suitable for PCR reaction.

3. The modification of virus

The sequencing results confirmed that VTT contains three target mutation sites, indicating successful viral modification.

4. The phagocytic plaque assay

1. Among them, wells 4 and 5 showed better results.

2. Well 4 has 33 plaques; well 5 has 6 plaques.

3. After calculation, the virus stock concentration is 3.67 x 10^5 pfu/mL.

Introducing an artificial translation system

The plasmid pcpAzpaRS_v2 was purchased through the Addgene platform. pcpAzpaRS_v2 can simultaneously transcribe tRNA in mammalian cells (293T) with human modifications at both the 3' and 5' ends, and translate EcTyrRS(Y37L/D182S/F183M/L186A). Additionally, it includes RNA polymerase III.

To enable HEK 293T cells to express both the folate receptor and mCherry, we will construct the plasmid in two steps. The first step is to introduce the genes for the folate receptor and mCherry into the plasmid. The second step involves inserting a T2A sequence between the folate receptor gene and the mCherry gene to prevent the expression of a fusion protein during recombinant vector expression, which could lead to experimental failure.

1. The construction of the recombinant expression vectors p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry

Introduce the genes for the folate receptor and mCherry into the plasmid.

2. The construction of the recombinant vector p3×FLAG-CMV-FOLR1-T2A-mCherry and recombinant vector p3×FLAG-CMV-FOLR2-T2A-mCherry

Inserting a T2A sequence between the folate receptor gene and the mCherry gene to prevent the expression of a fusion protein during recombinant vector expression, which could lead to experimental failure.

The sequencing results indicate that the plasmid modification was successful, and we successfully inserted a T2A sequence between the folate receptor gene and the mCherry gene.

3. The expression of FOLR1, FOLR2, and mCherry in HEK 293T cells

To enable HEK 293T cells to simultaneously express both the folate receptor and mCherry.

The plasmid FolR2-T2A-mCherry was successfully transfected and can be used for subsequent validation. The transfection of FolR1-T2A-mCherry failed, and the possible reason may be that the plasmid concentration is too low.

Using click chemistry to connect viruses with desired molecules & Testing whether the virus has been successfully modified

The virus express A27L-3stop & the click chemistry reaction between A27L-3stop and folate-PEG-DBCO , resulting viruses with targeted specificity

As can be seen from the figure, there is a significant amount of yellow resulting from the overlap of red and green fluorescence, indicating a good effect. This confirms that 4-L-azidophenylalanine has been used to synthesize A27L-3stop, resulting in better viral targeting.