Overview

Both norovirus and rotavirus are pathogens that cause diarrhea in infants and young children ¹ .   Vaccination is the primary means of preventing and controlling rotavirus and norovirus infections ² . Rotavirus vaccines have been clinically used for many years and can significantly alleviate symptoms caused by rotavirus infections ³ . Due to the difficulty in achieving in vitro isolation and cultivation of norovirus, there is currently no norovirus vaccine available for use. This study is mainly based on genetic engineering vaccines. We mainly used genetic engineering methods to construct corresponding plasmids to develop specific vaccines against norovirus  and rotavirus   viruses .

 

Results of Experiments

We will introduce the results of our project from the following four  aspects :

(1)  Construction of recombinant plasmids

(2)  Protein expression and purification

(3)  Expression optimization

(4)  Verification of protein expression in E. coli Nissle 1917

 

1. Construction of recombinant plasmids

To  construct three recombinant plasmids , i.e. ,   pGEX-GII.4-VP1 , pGEX-GII.17-VP1,  and pGEX- RV- GII.17-VP1, firstly, we amplified the GII.4-VP1 ( 1620 bp ) , GII.17-VP1 ( 1620 bp )  and RV - VP7  ( 843 bp )  fragments by PCR. As shown in F igure 1A, the three genes were amplified successfully compared to the 15K maker.  T hrough the double enzyme digestion experiment, we successfully obtained the linearized  plasmid pGEX-4T-1  ( Figure 1B ) , which was then ligated with GII.4-VP1 , GII.17-VP1  and RV - VP7 fragments , respectively. The mappings of recombinant plasmids after ligation are shown in Figure 1C-D.

 

Figure 1 Construction of three   plasmids

 

Subsequently the plasmids pGEX-GII.4-VP1 and pGEX-GII.17-VP1 were transformed into  E. coli  BL21 (DE3) competent cells, respectively. The plasmid pGEX- RV- GII.17-VP1 was  transformed into  E. coli  BL21 (DE3) and E. coli   Nissle 1917 , respectively. After overnight incubation, transformants were successfully grown on LB plates ( Figure 2 A-B). We performed colony PCR identification of the transformants, and the results are shown in the figure, about 2/3 of the transformants containing pGEX-GII.17-VP1 had the correct bands; all of the transformants containing pGEX-GII.4-VP1 or pGEX-RV-GII.17-VP1 had the correct bands ( Figure 2 C-D). To further verify the construction of the plasmid, we sequenced the positive transformants, and the sequencing results showed that the expression frame sequences were all correct, indicating that the plasmids were successfully constructed ( Figure 2 E-G).

 

Figure 2 Results of plasmid transformation, identification ,  and sequencing

 

2. Protein expression and purification

We inoculated positive transformants and induced protein expression by IPTG. After obtaining protein lysates, we purified the target proteins 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, but the purified protein was barely visible, which might be due to the low expression of protein. In the future, we plan to increase the expression level of the GII.4-VP1 protein. As shown in Figure 4, we successfully expressed and purified GII.17-VP1 protein ( 60  kDa).

            

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

(P represents " Precipitation" , S represents " Supernatant" , T represents " Flow through" , W represents " Wash solution" and E represents " Eluent " )

 

           

Figure 4 SDS-PAGE results of pGEX-GII. 17 -VP1  protein expression and purification

(P represents " Precipitation" , S represents " Supernatant" , T represents " Flow through" , W represents " Wash solution" , and E represents " Eluent " )

 

3.  Expression optimization

In addition to constructing norovirus-associated plasmids, we constructed recombinant plasmids incorporating norovirus and rotavirus antigenic genes. Similarly, 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 6A ). In addition, we examined the expression of the target proteins using SDS-PAGE ( Figure 5 ) and used the ImageJ software to quantify the target band  (119 KDa) 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 6B).

As shown in Figure 6, 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 5 SDS-PAGE results of RV- GII. 17 -VP1  protein under different expression conditions

 

Figure 6 Effect of IPTG induction time and bacterial concentration on protein concentration

 

4.  Verification of protein expression in   E. coli Nissle 1917

E. coli Nissle 1917 (EcN) is a Gram-negative probiotic and an ideal expression vector for oral live vaccines ⁴ . So 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 119   kD a  range, which means that RV- GII.17-VP1  protein expression level is weak in E. coli Nissle 1917  (Figure 7) .  This initially confirmed that RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be optimized.

 

Figure 7 SDS-PAGE results of pGEX- RV- GII. 17 -VP1  protein expression in E.coli Nissle 1917

(P represents " Precipitation" , S represents " Supernatant" , T represents " Flow through" , W represents " Wash solution" and E represents " Eluent " )

 

Future plan

At present, we have determined the optimal conditions for protein expression in E .  coli . Next, we will improve and optimize the protein expression method to achieve large-scale expression of RV- GII. 17 -VP1 in E. coli Nissle 1917 . In addition, the next stage of our project will be to obtain transformed   E. coli Nissle 1917  through tube feeding in mice, and then measure the levels of anti-RV-GII.17-VP1 IgG in mouse serum and IgA in feces. Finally, we can verify the immunogenicity of our oral vaccine.

 

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).

4.  Jacobi, C. A., & Malfertheiner, P. (2011). Escherichia coli Nissle 1917 (Mutaflor): new insights into an old probiotic bacterium. Digestive diseases (Basel, Switzerland), 29(6), 600-C607.