Results

Part1 Construction of Mono-MSP

1 MSP Plasmids Construction

In order to successfully construct nanodiscs, we decided to use the Membrane Scaffold Proteins (MSPs) that have been reported in the literature for initial trials and validation. By reviewing the literature, we decided to first use four types of monomeric MSPs, namely spNW50, spMSP1D1, spNW15, and MSP1E3D1, to manufacture nanodiscs.

We first synthesized the following plasmids at GENEWIZ, and the company returned the plasmids to us via transformation.

Then, we isolated single colonies using the streak plate method.

Lastly, through PCR method, we confirmed that the target genes had been obtained.

2 Cultures expansion and inducible expression

We performed amplification and cultivation of the obtained target colonies separately, and induced with IPTG when the OD600 value reached 0.6-0.8.

The concentration of IPTG and the induction time can both have a certain impact on protein expression levels. We conducted a brief exploration of the induction concentration and time for spNW50 and MSP1E3D1. We added different amounts of IPTG solution to the bacterial solutions, set the concentration gradient of the final concentration of IPTG, and selected the most appropriate concentration of IPTG. The most suitable IPTG concentration was screened by SDS-PAGE gel electrophoresis.

For spNW50, it was found that for the same induction time (10h), there was little difference in protein amount under different IPTG concentration(Figure 3c).

According to the electrophoretic results (Figure 3d), MSP1E3D1 bands were most obvious when the IPTG concentration was 0.8mM, and the IPTG concentration of 0.8mM was selected.

What's more, for spNW50, considering that induction time may have a certain effect on protein expression, we set two time gradients to explore the optimal induction time.

It was found that for the same IPTG concentration, there was little difference in protein spNW50 amount under different induction time (Figure 3c).

We used shaking tables for inducible expression under the induction conditions shown in the table below (Table 5). SDS-PAGE was used to analyze whether the induction was successful (Figure 3)

3 Protein expression and purification

The bacteria were broken up in the metal bath, and the crude protein extract was obtained. SDS-PAGE analysis was performed to test whether the induced expressions were successful.

3.1 spNW15

The theoretical size of spNW15 was 40.3kDa, and the crude protein extract after induction had obvious bands in the 40kDa attachment, which suggested that the expression of spNW15 was successfully induced (Figure 4a).

The bacteria were ultrasonically broken in Buffer A, and the proteins were refined by elution with nickel bead affinity chromatography and imidazole with gradient concentration. We conducted SDS-PAGE analysis to check the purification effect (Figure 4b). It can be seen that a large number of target proteins were purified in 300mM imidazole eluant and 500mM imidazole eluant, but the purity was not high and there were some impurity bands, which may be caused by the dimerization or polymerization of spNW15, and further purification was required through molecular sieve.

In order to obtain higher protein purity, we purified the protein using molecular sieve chromatography, and obtained spNW15 protein with higher purity through SDS-PAGE analysis (Figure 4c).From the results of SDS-PAGE, it can be seen that SEC was effective, and our spNW15 was purified. Subsequently, ultrafiltration tubes were used to concentrate and measure the concentration, so as to prepare the nanodiscs.

3.2 spMSP1D1

The theoretical molecular weight of spNW50 is 123.9 kDa. Compared with the liquid phase of crude protein extracts without induction, there is a distinct band obtained in the crude protein extract after induction, indicating that the induction expression of spNW50 was successful (Figure 6a).

The bacteria were ultrasonically broken in Buffer A, and the proteins were refined by elution with nickel bead affinity chromatography and imidazole with gradient concentration. SDS-PAGE analysis was performed to check the purification effect (Figure 5b). It can be seen that a large number of target proteins were purified from 100mM imidazole and 300mM imidazole elution, but the purity was not high and there were some impurity bands, which may be caused by dimerization or polymerization of spMSP1D1. Further purification by molecular sieve was required.

In order to obtain higher purity proteins, molecular sieve chromatography was used to purify proteins. After SDS-PAGE analysis (Figure 5c), we obtained high purity spMSP1D1 and suspected spMSP1D1 dimer proteins, which were then concentrated and measured by ultrafiltration tube for subsequent preparation of nanodiscs.

What's more, we further used anti-His antibodies and confirmed the successful purification of spMSP1D1 protein through Western Blot (Figure 5d). Additionally, it has been determined that what was previously suspected to be spMSP1D1 dimers may not be dimers but rather non-specific bands.

3.3 spNW50

The theoretical size of spNW50 was 123.9kDa. Compared with the liquid phase of uninduced crude protein extraction, the induced crude protein extract showed more obvious bands in the range of 135-180kDa, which suggested that the induced expression of spNW50 was successful (Figure 6a).

A cracking buffer containing 0.02%Triton x 100 was used and ultrasonically broken, followed by nickel strain elution for fine protein purification. SDS-PAGE analysis was performed again to verify the purification effect (Figure b). Obvious bands can be seen, and there are few impurity bands. So it is considered that we have purified relatively pure spNW50.

The protein concentration was determined using the Bradford method (see protocol for details) to facilitate subsequent fabrication of the nanodiscs.

3.4 MPS1E3D1

The theoretical size of MSP1E3D1 was 32.6kDa. Compared with the liquid phase of uninduced crude protein extraction, the induced crude protein extract showed more obvious bands in the range of 25kDa to 35kDa, suggesting that MSP1E3D1 was successfully induced. Moreover, it was observed that the MSP bands were more obvious when the IPTG concentration was 0.8mM, so the optimal induced concentration of IPTG was 0.8mM (Figure 7a).

A cracking buffer containing 0.02%Triton x 100 was used and ultrasonically broken, and then nickel strain eluted to refine the protein. SDS-PAGE analysis was performed again to verify the purification effect (Figure 7b). Obvious bands were visible at 25-35kDa in the lane of the sample eluant, and there were few impurity bands, so it was considered that we purified relatively pure MSP1E3D1.

MSP1E3D1 was further purified by molecular sieve (Figure 7c). The purification effect was tested by SDS-PAGE analysis (Figure 7d). A shallower band can be seen at 25-35kDa in the lane of Peak 1, presumed to be purified MSP1E3D1, but at a lower concentration.

MSP1E3D1 purified by molecular sieve was concentrated by ultrafiltration tube, and then the purification success was verified by SDS-PAGE (Figure 7e). There was a relatively obvious band at 25-35kDa of the swim lane of MSP1E3D1, which was presumed to be the concentrated MSP1E3D1, indicating successful purification.

Ultimately, Bradford method (Figure 6c) was used to determine the protein concentration of the above four proteins (see protocol for details) to facilitate subsequent fabrication of the nanodiscs.

Part2 Construction of Multi-Polymerized MSP

We aim to utilize different linkers (Spy/Sdy/Snoop) to connect Mono-MSPs to form multi-polymerized MSP, thus creating larger nanodiscs. Larger nanodiscs are advantageous for accommodating more receptor proteins and can increase the probability of nanodisc-virus binding in vivo. Compared to directly producing longer MSP proteins, this method effectively alleviates the metabolic stress on the bacteria. Additionally, through the fluorescence emitted by mCherry, we can effectively validate our idea.

1 Plasmid Construction

To produce polymeric MSPs, we constructed plasmids to synthesize three MSPs separately, in order to achieve controllable cyclization of MSP and controllability of the nanodisc particle size. In the experiments of the previous section, we purified MSP proteins of different lengths to construct nanodiscs of varying sizes. However, the purification of proteins with a large molecular weight (such as spNW50) presented certain difficulties, thus further illustrating the necessity of our newly designed method for constructing larger nanodiscs (Multi-polymerized MSP).

We obtained the gene sequence of spNW15 from NCBI and integrated the sequences of the three linkers (Spy/Sdy/Snoop) in a specific order at its N-terminus and C-terminus to form three MSPs that can be connected to each other. Based on the different linkers fused at both ends of the MSPs, we sequentially name these fusion proteins used to generate multi-polymerized MSP as SCSdC-mCh[1-10], SnCSdT, and SnTST-mCh[11]. We added 5' (NcoI) and 3' (XhoI) to the ends of the genes (BBa_K5301005, BBa_K5301006, BBa_K5301007) through GENEWIZ, cloning them into the vector pET-28a(+) (Kanamycin) to construct three recombinant plasmids, which were then introduced into BL21 (DE3). Subsequently, we picked multiple single colonies from the plate for colony PCR to check if the plasmids were successfully introduced into the host bacteria. We selected colonies with the desired bands for the next step of induced expression.

2 Induced Protein Expression

We selected suitable single colonies and cultured them overnight in 20 mL LB (Kanamycin). The overnight culture was then added to fresh 200 mL LB (Kanamycin) at a 1:100 ratio and incubated at 37°C for approximately 3 hours (slightly less than 3 hours, depending on the concentration of the activated bacterial liquid). When the culture reached an OD600 of 0.6-0.8, we induced protein expression with IPTG and continued incubation at 16°C for 16 hours. For the SCSdC-mCh[1-10] and SnCSdT, we chose a final IPTG concentration of 0.8 mM. After observing poor expression of the SnTST- mCh[11] in the multi-polymerized MSP, we set an IPTG concentration gradient to find the optimal IPTG concentration for protein expression. The bacterial culture was then centrifuged, and the pellet was subjected to SDS-PAGE to compare protein expression levels under different IPTG concentrations, identifying the optimal induction concentration as 1 mM.

3 Protein Extraction and Purification

3.1 Protein Purification

After determining the optimal induction conditions for the three proteins, we scaled up the culture. We incubated the strains producing SCSdC-mCh[1-10], SnCSdT, and SnTST- mCh[11] at 16°C with IPTG concentrations of 0.8 mM, 0.8 mM, and 1 mM, respectively, for 16 hours, thus obtaining a large amount of target protein.

In the plasmid design, we included a His-tag in the target proteins to facilitate the use of nickel affinity chromatography for obtaining high-purity target proteins. As shown in the figure, the use of 300 mM and 500 mM imidazole successfully eluted a substantial amount of SCSdC-mCh[1-10] and SnCSdT

Due to the potential interference of endogenous E. coli proteins and possible dimerization or degradation of MSP proteins with SDS-PAGE, we did not verify the successful purification of SnTST- mCh[11] through SDS-PAGE. We further used anti-His antibodies and confirmed the successful purification of SnTST- mCh[11] through Western Blot.

Due to the nickel column purification results not meeting the expected protein purity, we further separated and obtained high-purity SCSdC-mCh[1-10] through SEC, as shown in the figure.

3.2 Protein Concentration Determination

To facilitate the subsequent connection of multi-polymerized MSPs and the construction of large cyclic nanodiscs, we used the Bradford method to plot a standard curve and measured the concentration of SCSdC-mCh[1-10] protein to be 0.228 mg/ml.

4 Structure and Function Validation

When constructing multi-polymerized MSP, We predicted and analyzed the structure of three fragments and the overall structure of the multi-polymerized MSP using AlphaFold2, to confirm that the structure of the fusion protein is rational.

By controlling the reaction conditions, we successfully connected the three segments of MSP proteins. To characterize the connection of the large cyclic MSP, we used SDS-PAGE to verify the molecular weight of the multi-polymerized MSP and observed the fluorescence of mCherry under a fluorescent inverted microscope to characterize the successful cyclization of the MSPs. Although the SDS-PAGE results are not clear (for specific content, please refer to the engineering section), clear fluorescence can be seen under the fluorescence microscope (Figure 17).

In future experiments, we hope to further characterize multi-polymerized MSP to confirm its successful polymerization. Due to time constraints, we have only used Mono-MSP to produce nanodiscs and validate the Nanodisguiser function thus far. In upcoming experiments, we aim to use multi-polymerized MSP to manufacture nanodiscs and insert membrane proteins to better achieve the functionality of Nanodisguiser.

Part3 Membrane Protein Polymerization

1 Construction and Verification of Plasmid Vectors

1.1 Construction of Plasmid Vectors

To obtain Monomeric Streptavidin (mSA) fused with split-GFP, we designed two plasmids: pET-22b-mSA-sGFP1-10 and pET-22b-mSA-sGFP11, where mSA-sGFP1-10(or sGFP1-10 tether BBa_K5301017) and mSA-sGFP11 (or sGFP11 tether BBa_K5301018) are our target fragments. To enhance protein expression, the sequence was optimized for codon usage.

1.2 PCR Verification

After isolating single colonies on solid media, different colonies were picked, and colony PCR was performed to verify whether the plasmid was successfully transformed.

The target fragment (BBa_K5301017) length is 1071 bp, and all colonies successfully obtained the target fragment.

The target fragment (BBa_K5301018) length is 477 bp, and colonies 1, 2, 3, 5, and 6 successfully obtained the target fragment.

Colonies with successfully transformed plasmids were inoculated into 20 mL of LB liquid medium for single strain culture.

2 Expression Induction and Protein Purification

2.1 Screening of Expression Induction Conditions

The presence of mSA in sGFP1-10 and sGFP11 tethers often leads to the formation of inclusion bodies, increasing purification difficulty. After reviewing the literature, we found that low-temperature expression can reduce inclusion body formation, so we determined 16°C as the induction temperature. To achieve more efficient expression of sGFP1-10/sGFP11 tethers, we set up a gradient of IPTG concentrations: 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM. The results indicated that protein expression of sGFP1-10 tether reached its optimum at 0.75 mM, while sGFP11 tether reached its optimum at 0.5 mM.

2.2 Protein Purification

We used 1 mM IPTG to induce the strain for expressing the target protein and extracted the protein after inducing for 16 hours at 16°C. During the plasmid construction process, we incorporated a His-tag into the target protein to facilitate purification using nickel column affinity chromatography based on His-tag-specific binding. In this section, we explored the conditions repeatedly, which will be described in detail in the engineering section. Here, we present some of our successful results.

The molecular weight of sGFP1-10 tether is 39.2 kDa, while that of sGFP11 tether is 17.0 kDa. After purifying the supernatant obtained from the lysate of the bacterial culture, we found that the amount of soluble protein was low, and the protein existed in inclusion bodies in the precipitate. Therefore, we simultaneously purified the supernatant and inclusion body proteins, sequentially using elution buffers containing imidazole at concentrations of 100 mM and 150 mM to elute the proteins. The SDS-PAGE results, as shown in the figures, indicated that sGFP1-10 tether was eluted more effectively with the 150 mM elution buffer, while sGFP11 tether showed better elution with the 100 mM elution buffer. Gel analysis of the nickel beads revealed that a significant amount of sGFP11 tether was not eluted.

To improve the elution of proteins from the nickel beads, we attempted to increase the imidazole concentration, sequentially using elution buffers containing imidazole at concentrations of 100 mM, 250 mM, and 500 mM.It can be observed that increasing the imidazole concentration results in a larger amount of eluted protein. However, a higher imidazole concentration is not necessarily better; the elution buffer with 250mM imidazole eluted the highest amount of protein.

2.3 Protein Concentration

We determined the protein concentration using the Bradford method, a dye-binding assay. Coomassie Brilliant Blue is red in its free state, with a maximum absorbance at 488 nm; upon binding to protein, it changes to blue, and the protein-dye complex has maximum absorbance at 595 nm. The absorbance value is proportional to the protein content, making it suitable for quantitative protein analysis. The binding of protein to Coomassie Brilliant Blue reaches equilibrium in about 2 minutes, and the reaction completes rapidly; the complex remains stable at room temperature for up to 1 hour.

1. NanoDrop

First, we prepared a gradient standard of BSA and prepared protein samples, using NanoDrop to determine the standard curve and measure protein concentrations. The results are as follows. The R² value of the standard curve for this measurement was 0.928, indicating a good fit. The measured concentrations were 81.540 μg/mL for sGFP1-10 tether and 81.540 μg/mL for sGFP11 tether.

2. Microplate Reader

We prepared a gradient standard BSA solution and sample, using a microplate reader to determine the standard curve and measure protein concentrations (note: this batch of protein is different from that measured by NanoDrop). The gradient standard BSA solution was added to wells B1 to B11 of a 96-well plate, while the protein samples were added to wells C1 to C6, D1 to D8, and E1 to E8. The BSA standard curve is shown in Figure 11, and the measured protein concentrations are presented in Table 1.

3 Protein Activity Analysis

In the process of purifying inclusion body proteins, we used a high concentration of urea for denaturation and solubilization. To ensure that streptavidin protein and split-GFP retain biological activity in subsequent conjugation experiments, we refolded the proteins. To determine the effectiveness of refolding, we measured the activity and concentration of the sGFP1-10 / sGFP11 tether proteins, using a method based on the ELISA assay.

Specifically, this experiment utilized the strong affinity between streptavidin and biotin, using HRP (horseradish peroxidase) as the labeled enzyme and biotin as the linking molecule. The enzymatic activity of HRP causes a color change in the substrate TMB, and the absorbance was measured using a microplate reader to assess the activity of streptavidin.

Due to some experimental errors, we were unable to obtain the mSA standard curve. Therefore, it is not possible to obtain specific protein activity data. But we have judged the protein activity through direct observations. By comparing the absorbance values with those of the mSA standards and the 0.5% BSA negative controls, it can be observed that the protein activity obtained from the inclusion body purification is very low. Even after the renaturation process, the activity did not improve. In contrast, the protein purified directly from the supernatant exhibits some activity and can be used for subsequent ligation experiments.

4 Biomolecular Fluorescence Complementation(BiFC)

We first investigated the incubation conditions for the binding of sGFP1-10/sGFP11 tether by mixing solutions of sGFP1-10 tether protein and sGFP11 tether protein in various volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. After incubation at 4°C, we observed whether fluorescence appeared.

We observed fluorescence in some of the mixed incubation samples, indicating that the sGFP1-10 tether and sGFP11 tether successfully bound. Next, we will change the concentration ratio of sGFP1-10/11 tether, incubation time, etc. to explore the optimal conditions for their binding. Meanwhile, we will analyze the possible reason for the weak fluorescence due to low protein concentration, so we will also continue to optimize the method of purifying sGFP1-10/11 tether protein.

5 Binding of Biotinylated Membrane Proteins

In subsequent experiments, we will proceed with the purification and activity validation of sGFP1-10/11 tether, and simultaneously conduct BiFC (Biomolecular Fluorescence Complementation) experiments. We will utilize GFP handles to purify the successfully bound sGFP1-10/sGFP11 tether dimers. Once sufficient dimers are obtained, we will biotinylate the membrane proteins purchased with avi-tags and link them through the streptavidin-biotin system, ultimately constructing membrane protein dimers.

Part4 Assembly of Nanodiscs

1 spNW15

1.1 Native-page:

We prepared nanodiscs with different lipid-to-protein ratios, and immediately performed Native-PAGE characterization after the preparation was completed. Compared with lane 6, lane 4 (1:100 spNW15) had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful (Figure 30).

1.2 Negative Stain Electron Microscope(EM)

We prepared negative stain electron microscope samples using the nanodiscs from lane 4 (1:100 spNW15) in Native-PAGE and performed negative stain electron microscopy imaging. The results are shown below (Figure 31). In the image, we can see that the black circular objects are the nanodiscs. Their diameter is approximately 15 nm, which is close to the theoretical value.

2 spMSP1D1

2.1 Native-PAGE

We prepared nanodiscs with different lipid-to-protein ratios, and immediately performed Native-PAGE characterization after the preparation was completed. Compared with lane 1, other lanes had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful (Figure 32a).

Next, we will prepare spMSP1D1 nanodiscs incorporating the membrane protein LDL-R and perform preliminary validation using Native-PAGE (Figure 32b). We prepared nanodiscs with a LDL-R:spMSP1D1:DOPC ratio of 2:5:60 and 1:2:130. First, the membrane protein is mixed with DOPC and incubated on ice for 1 hour to form lipid-detergent micelles. Then, spMSP1D1 protein is added to the protein-lipid mixture and incubated for 10 minutes. Fresh Bio-beads are then added to adsorb the detergent from the mixture, inducing the self-assembly of nanodiscs. The mixture is gently incubated at 4°C for 2 hours before being supplemented with fresh Bio-beads and continuing to incubate overnight at 4°C with gentle shaking.

2.2 negative stainEM (negative stain electron microscope)

We prepared negative stainEM samples using the spMSP1D1 nanodiscs and performed negative stain electron microscopy imaging. The results are shown below (Figure 33a). In the image, we can see that the black circular objects are the nanodiscs. Their diameter is approximately 6-8 nm, which is close to the theoretical value of 11nm (Figure 33c). Surprisingly, we also observed the nanodiscs from the side (Figure 33b). We also used negative stain EM to observe the nanodiscs incorporating LDL-R (Figure 33e). In the images, some black dots can be seen on the nanodiscs, which we believe are the membrane proteins.

3 spNW50

3.1 Native-PAGE

The lipid-to-protein ratios had been used of 1:600, and immediately performed Native-PAGE characterization after the preparation was completed. Compared with lane and 5, lane 2 and 3 had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful.(figure 34)

3.2 DLS (Dynamic Light Scattering) &negative stainEM (negative stain electron microscope)

We prepared DLS samples using the nanodiscs from lane 3 in Native-PAGE and performed the DLS. The result showed that, the diameter of nanodiscs was around 300nm (figure 35d.)

Then we prepared negative stain electron microscope samples using the spNW50 nanodiscs and performed negative stain electron microscopy imaging. The results are shown below (Figure 35abc). In the image, we can see that the black circular objects are the nanodiscs. Their diameter is approximately 300 nm, which is close to the DLS result.

Part5 Functional Verification

1 Construction of the three plasmids

In the part of functional verification, we established a new SEND system to simulate the process of virus invading cells via membrane fusion. The novel SEND system demands three plasmids: capsid, cargoRNA, and fusogen. The capsid and fusogen plasmids we utilized were sourced from the commercial offerings of Miaoling Biotechnology. We transferred the pre - made plasmid products into E.coli DH5α for amplification and extraction.

Regarding the cargoRNA plasmid, we joined the commercial cargo backbone to the superfolderGFP fragment through homologous recombination. The superfolderGFP fragment was obtained from an official plasmid donated by iGEM. For the effective function of cargoRNA, the reporter gene (superfolderGFP) needs to be connected on both sides of PEG105'UTR and PEG103'UTR; thus, we opted for seamless cloning of the plasmid. Firstly, we amplified and extracted the cargo backbone plasmid and superfolderGFP plasmid. Subsequently, we obtained a linearized cargo backbone (carrying PEG10 UTR sequences on both sides) by reverse PCR. Meanwhile, the superfolderGFP fragment sequence was procured from the plasmid by PCR. To obtain the two pure linear PCR products, we employed agarose gel for slice recycling. Finally, using an infusion kit, we constructed the recombinant cargoRNA plasmid. To affirm the validity of the plasmid, we carried out fragment sequencing. All the experimental data indicated that the recombinant cargoRNA plasmid was successfully constructed.

2 HEK 293FT cell culture

The cells used in the verification experiment were HEK 293FT cells from Shanghai Binsui Biotechnology. HEK 293FT cells are adherent cells with a polygonal epithelial-like appearance. Through microscopic observation during multiple passages, the cell status and viability were confirmed.

3 VLPs obtainment after plasmid transfection

To confirm the successful transfection of the three plasmids and the effective expression of VLP, we conducted SDS - PAGE verification. The results of SDS-PAGE showed that the cells produced proteins similar in size to PEG10 and VSV-G. However, protein bands also appeared at the same positions in the control medium and in the supernatant of normal cells. (Fig 40B.) To further verify the successful transfection of the plasmids, fluorescent monitoring of the cells was performed. We observed that 24 hours after transfection, the cells emitted green fluorescence. (Fig 40A.) And these results suggest that the transfected cells may have expressed exosomes containing PEG10 VLPs.

4 Cells infected by VLPs

In order to confirm that PEG10 VLP exosomes are capable of infecting target cells and delivering mRNA, and to verify the blocking effect of nanodiscs on VLP, we performed a controlled infection experiment on HEK293 FT cells. The infusions added to the cells of the experimental group, positive control group and negative control group were successively: VLP supernatant containing nanodiscs, VLP supernatant alone, and cell culture medium containing neither VLP nor nanodiscs. After adding the corresponding solution, fluorescence monitoring was performed on the cells at an interval of 30 minutes. In the observation of the positive control group, we found that the internal cells did emit green fluorescence. This indicates that the cells acquired the mRNA of the GFP protein and expressed it. The only possible source of this mRNA is the supernatant we added. This result confirms that the novel SEND system can produce PEG10 VLP exosomes capable of effectively transporting mRNA, leading to its expression in target cells.

Furthermore, fluorescence observations showed that the cells with only culture medium did not express fluorescence. And on the whole, cells treated with VLPs alone expressed more GFP protein compared to those that also received nanodiscs. (Figure 42) This result partially validates the blocking effect of nanodiscs on the pseudovirus VLPs. It is speculated that the nanodisc has a blocking effect on membrane fusion virus.

5 Further Experiments

In the final control infection experiment, we were unable to obtain further fluorescence intensity data due to time constraints. Counting fluorescent cells by eye can introduce certain errors. Additionally, the monitoring of fluorescence levels during the experiment may have been influenced by the selection of the microscopic field of view. Our conclusions may be affected by these factors, so we plan to conduct more rigorous validations in future experiments, such as fixing the observation field and obtaining fluorescence intensity values to reflect infection rates. For conclusions based on indirect evidence, we aim to design more direct experiments. For example, we plan to use Western Blot and electron microscopy to directly confirm the expression of PEG10 VLP exosomes. Regarding the verification of the blocking function of nanodiscs on VLPs, we hope to use methods like DLS to provide a more microscopic perspective on the membrane fusion process.