Introduction:  

Norovirus is characterized by high infectivity, diverse modes of transmission, and easy mutation of the pathogen, and is the main pathogen of sporadic and fulminant acute gastroenteritis in the population, which causes a huge disease burden to the society, and there is no vaccine for norovirus in the market at present ^1-3 . Therefore, our team is attempting to produce a vaccine for norovirus, and meantime we also tried to produce bivalent vaccines against norovirus and rotavirus .

In this process, we first genetically engineered the major antigenic genes of norovirus or (and) rotavirus into vector plasmids and transformed them into E. coli  BL21(DE3) or Nissle 1917 , and then induced the plasmids to express the proteins upon culture. Finally, we purify and analyze the proteins.

 

Cycle I : BBa_K4872010  ( pGEX-GII. 4- VP1)  

Design:

We used the Tac promoter, which is suitable for E. coli protein production, for transcription of the expression frame. At the C-terminus of target gene GII.4-VP1, we fused a GST tag, which facilitates soluble expression of the protein and can be used to purify the protein (Figure 1). After obtaining this recombinant plasmid, we transformed it into  E. coli BL21(DE3).   Finally, we used IPTG to induce the expression of the target protein GII.4-VP1.

 

Figure 1 Plasmid design diagram  of pGEX-GII.4-VP1

 

Build:

Firstly,  we  amplified  the antigen gene GII.4-VP1 us ing  the PCR amplifier   ( Figure 2A ). Then,   the GII.4-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with restriction endonucleases Eco RI and Xho I, followed by the ligation using T4 DNA ligase to obtain the recombinant plasmid pGEX-GII.4-VP1 (Figure 2B) . As shown in Figure 2C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

 

Figure 2 Construction of plasmid pGEX-GII.4-VP1

 

Test:

We inoculated the positive transformant and induced protein expression by IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein expression and purification results by SDS-PAGE. The results are shown in Figure 3, we successfully expressed GII.4-VP1 protein  (60 KDa) , but the purified protein was barely visible, which might be due to the low expression of protein.

            

Figure 3 SDS-PAGE results of pGEX-GII.4-VP1  protein  expression

(P represents " Precipitation" , S represents " Supernatant" , Trepresents " Flow through" , Wrepresents " Wash solution" and Erepresents " Eluent " )

 

Learn:

Since the low expression of the protein was not successfully purified, we need to explore the optimal expression conditions for the purification of this protein to increase the expression level of the protein.  After we solve this problem, we could further step to continue the construction of recombinant plasmid pGEX-RV VP7-GII.4-VP1.

 

Cycle2: BBa_K4872011  ( pGEX-GII.17-VP1 )

Design:

Following the same idea as the previous round, we used the Tac promoter to transcribe the plasmid A GST tag was fused to the C-terminus of the target gene GII.17-VP1 (Figure 4). After obtaining the recombinant plasmid, we transformed it into E. coli  BL21(DE3). Finally, we used IPTG to induce the expression of the target protein GII.17-VP1.

 

Figure 4 Plasmid design diagram of pGEX-GII.17-VP1

 

Build:

W e  first amplified  the antigen gene GII.17-VP1 us ing  the PCR amplifier ( Figure 5 A ). Then,   the GII.17-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with restriction endonucleases Eco RI and Xho I, followed by the ligation using T4 DNA ligase to obtain the recombinant plasmid pGEX-GII.4-VP1 (Figure 5 B) . As shown in Figure 5C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

 

Figure 5 Construction of plasmid pGEX-GII.17-VP1

 

Test:  

We inoculated the positive transformant and induced protein expression by IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein expression and purification results by SDS-PAGE. As shown in Figure 6, we successfully expressed and purified GII.17-VP1 protein (60 kDa).

 

             

Figure 6 SDS-PAGE results of pGEX-GII.17-VP1 protein expression

(Prepresents " Precipitation" , Srepresents " Supernatant" , Trepresents " Flow through" , Wrepresents " Wash solution" and Erepresents " Eluent" )

 

Learn:

Unlike the expression and purification of GII.4-VP1 protein, the GII.17-VP1 protein was successfully expressed and purified in this round of experiments, which is expected to be prepared as a norovirus vaccine.  In order to develop this norovirus vaccine, the next plan will be focused on the research of the GII.17-VP1 expression in E. coli Nissle 1917 and more evaluation tests shall be made before the implementation. Meantime, we can continue to construct the recombinant plasmid pGEX-RV-GII.17-VP1 to investigate the potential of the bivalent vaccine against norovirus and rotavirus.

 

Cycle III: BBa_K4872013  ( pGEX-RV-GII.17-VP1 )

Design:

To obtain a bivalent vaccine against norovirus and rotavirus, we used a linker that connects the GII.17-VP1 antigenic gene of norovirus to RV VP7 of rotavirus. As in the previous two rounds of experiments, we used the Tac promoter and added a GST tag to the C-terminus of the fusion gene for subsequent purification (Figure  7 ). After obtaining the recombinant plasmid, we transformed it into E. coli  BL21(DE3) and Nissle 1917 , respectively. Finally, we used IPTG to induce the expression of target proteins and optimized their expression conditions.

 

Figure 7 Plasmid design diagram of pGEX-RV-GII.17-VP1

 

Build:

W e  first amplified  the antigen gene  RV VP7 and GII.17-VP1 us ing  the PCR amplifier ( Figure 8 A ). Then,   the RV VP7  and GII.17-VP1 fragments underwent homologous recombination with the pGEX-4T-1 plasmid vector to obtain the recombinant plasmid pGEX-GII.4-VP1 (Figure 8 B) . As shown in Figure 8C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

 

 

Figure 8 Construction of plasmid pGEX-RV-GII.17-VP1

 

Test:

We inoculated the transformant containing pGEX-RV-GII.17-VP1, induced its expression, and explored its optimal expression conditions.   To find the optimal conditions for the highest protein expression, we chose different concentrations (OD ₆₀₀ =0.3/0.6/0.8/1) of the bacterial solution and different IPTG induction times (0 h/4 h/8 h/16 h). After obtaining each protein lysate, we measured the total protein amount (A280) under different induction conditions ( Figure 10A ). In addition, we examined the expression of the target proteins  (119 KDa) using SDS-PAGE ( Figure 9 ) and used the ImageJ software to quantify the target bands on the SDS-PAGE gel, collected and organized the data, and plotted a line graph with OD ₆₀₀  as the x-axis and gray value as the y-axis (Figure 10B).

As shown in Figure 10, the protein concentration roughly tended to increase with increasing bacterial concentration at the same IPTG induction time. When the bacterial concentration was 0.8 and the induction time was 8 hours, the best protein expression level was achieved, indicating that this condition was more suitable for expressing more target proteins.

 

 

Figure 9 SDS-PAGE results of RV-GII.17-VP1 protein under different expression conditions

 

 

Figure 10 Effect of IPTG induction time and bacterial concentration on protein concentration

 

Finally, we transformed  plasmid pGEX- RV- GII.17-VP1  into E. coli Nissle 1917. Through the SDS-PAGE gel map, it was found that we have weak bands within the 90   kD a  range, which means that RV- GII.17-VP1  protein  (119 KDa)   expression level is weak in E. coli Nissle 1917  (Figure 11) .  This initially confirmed that RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be optimized.

 

Figure 11 SDS-PAGE results of pGEX-RV-GII.17-VP1 protein expression in E. coli Nissle 1917

 

Learn:

The expression of RV-GII.17-VP1 in E. coli Nissle 1917 was low, so we need to improve and optimize the protein expression method to achieve large-scale expression of RV-GII.17-VP1.  But we did see a potential of this bivalent vaccine against norovirus and rotavirus,  no doubt this research will be continually developed and is a promising product in the vaccine market for society.

 

References:

1.  Hallowell, B. D., Parashar, U. D., & Hall, A. J. (2019). Epidemiologic challenges in norovirus vaccine development. Human vaccines & immunotherapeutics, 15(6), 1279-C1283.

2.  Esposito S, Principi N. Norovirus Vaccine: Priorities for Future Research and Development.  Front Immunol. 2020 Jul 7;11:1383. 

3.  Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral immunology,2018,31(2).