Rotaviruses (RVs) in the family Reoviridae are the most common causative agents of severe gastroenteritis in children under five years of age, leading to significant morbidity and mortality worldwide, particularly in developing nations (Cohen et al., 2022). The RV virion is composed of an approximately 80 nm in diameter triple-layered protein capsid that encapsulates 11 segments of double-stranded RNA genome[1]. When observed under an electron microscope, rotavirus appears as a particle with short protrusions and a smooth outer edge, resembling a wheel shape (Figure 1).

Figure 1. The schematic diagram of rotavirus structure.

Before introducing vaccines for RV, most young children will likely be infected by RV at least once before age 5. After the vaccine was introduced, the efficacy in developed countries reached 80%-90%, yet the effectiveness significantly decreased in low- and middle-income countries by 40%. Though the cause of this drastic decline remains unclear, studies point out potential intestine-related issues, including coadministration of the oral poliovirus vaccine, malnutrition, and changes in microbiome composition. There are also safety issues related to intussusception, which is caused by the replication of bacteria in the intestine. To address these problems, nonreplicating subunit RV vaccine is urgently needed, as it can bypass the intestine system[2].Current subunit vaccines use trimers like Folden, which are non-human protein sources. Fusing them with the protein antigen can form a stable trimer, yet this will decrease the immune response.

In this study, we experimented with humanized type III collagen (partial sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPn of Rh3, a type III collagen, a homotrimer) or model collagen sequences (POGn, where O represents hydroxyproline) were used as novel trimeric motifs. The Vp4 antigenic trimer was formed using synthetic biology methods by fusion expression with the rotavirus Vp4 protein. Since prokaryotes lack proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)), and hydroxylation is essential for collagen stability, we co-expressed P4H derived from mega viruses with the Vp4 proteins described above to achieve stability of Vp4 oligomers.

Figure 2. Schematic representation of the Rh3/POG-conjugated VP4 design as a trimeric vaccine antigen.

Furthermore, we utilized to reported artificially designed proline-free heterotrimeric collagen-like protein motifs ((PKG)n, (DKG)n, (EPG)n), where three distinct serological VP4 proteins (P4, P8, P15) were fused to one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed in vitro to form oligomers. In this way, a multivalent-oligomeric rotavirus vaccine in E. coli is possible without P4H modification.

Figure 3. Schematic representation of PKG/DKG/EPG-conjugated VP4 design as a heterotrimeric vaccine antigen.

Part 1: BBa_K5531010 (pET-Dual-HisRh3C-P15VP4-P4H)

Design 1:

This targeted gene, Rh3C-P15VP4 (BBa_K5531000), synthesized by a biotech company, contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15. Meanwhile, P4H (BBa_K5531012) is also co-expressed in this plasmid due to the lack of proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)) in procaryotes, which is essential for collagen stability. The pET-Dual-N-His-TEV (BBa_K5531006) serves as the vector. The homologous recombination method was employed to construct pET-Dual-HisRh3C-P15VP4-P4H (BBa_K5531010).

Figure 4. The plasmid map of pET-Dual-HisRh3C-P15VP4-P4H

Build 1:

We isolated pET-Dual-N-His-TEV vectors from bacterial solutions primarily through centrifugation. The vectors were then recovered from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and proceeded to analyze the products through electrophoresis. The electrophoresis result displayed correctness toward the expected outcome (HisRh3C-P15VP4 is 2000 bp, and P4H is 755 bp). We selected several individual colonies for sequencing. Figure 5C shows the success in pET-Dual-HisRh3C-P15VP4-P4H construction.

Figure 5. The results of pET-Dual-HisRh3C-P15VP4-P4H

Test 1:

Later, the plasmid mounted with the gene of Rh3C-P15VP4 was transferred to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli BL21, which can help express His-Rh3C-P15VP4. After the colony PCR of E.coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression.

After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein Rh3C-P15VP4 has a size of 85 kDa (Figure 6).

Figure 6. The expression of His-Rh3C-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

Collagen-VP4 A was diluted to a concentration of 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 7, the samples exhibited two distinct clusters: high-molecular-weight and low-molecular-weight states, each displaying multiple bands indicating varying levels of proline hydroxylation.. Based on the construct design, we hypothesized that the high-molecular-weight clusters corresponded to trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 7. The SEC peaks for collagen-VP4 A ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimeric and monomer macromolecules.

Figure 7. Native-PAGE analysis (left)of oligomeric states of collagen A; Size exclusion chromatographic (SEC) analysis(right) of oligomeric states of collagen A.

Learn 1:

The first is that the activity of bacteria should be considered, as some of them failed to be cultured under the correct environment. The selection of primers and PCR temperature for different stages should be reevaluated, as the formation of primer dimers during PCR amplification may lead to inaccurate PCR results.

Part 2: BBa_K5531011 (pET-Dual-HisPPG-P15VP4-P4H)

Design 2:

The collagen sequence (POGn, O is hydroxyproline) was synthesized with the VP4 gene from the rotavirus with serotype P15. Meanwhile, P4H (BBa_K5531012) is also co-expressed in this plasmid due to the lack of proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)) in procaryotes, which is essential for collagen stability. The pET-Dual-N-His-TEV (BBa_K5531006) serves as the vector. The homologous recombination method was employed to construct pET-Dual-HisPPG-P15VP4-P4H(BBa_K5531011).

Figure 8. The plasmid map of pET-Dual-HisPPG-P15VP4-P4H

Build 2:

We isolated pET-Dual-N-His-TEV vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis result displayed consistency toward the expected outcome (HisPPG-P15VP4 is 2000 bp and P4H is 630 bp), indicating the success in pET-Dual-HisPPG-P15VP4-P4H construction. We selected several individual colonies for sequencing. Figure 9C shows the success of pET-Dual-HisPPG-P15VP4-P4H construction.

Fig 9. The results of pET-Dual-HisPPG-P15VP4-P4H

Test 2:

Later, the plasmid mounted with the gene of PPG-P15VP4 was transferred to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli BL21, which can help express His-PPG-P15VP4. After the colony PCR of E.coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression.

The protein expressed via E.coli BL21 was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis. The target protein PPG-P15VP4 has a size of 59 kDa (Figure 10).

Figure 10. His-PPGP15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

Collagen-VP4 A was diluted to a concentration of 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 11, the samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation. Based on the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 11. The peaks for collagen-VP4 B ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimer and monomer macromolecules.

Figure 11. Native-PAGE analysis (left)of oligomeric states of collagen B; Size exclusion chromatographic (SEC) analysis(right) of oligomeric states of collagen B

Learn 2:

It is crucial to consider the activity of bacterial strains when conducting experiments, as the initial activity of the strains plays a vital role in the entire experimental process. Proper preservation of the bacterial strains is essential to ensure the orderly progression of subsequent experiments.

Part 3: BBa_K5531007 (pET24a-DKG50-P15VP4)

Design 3:

This targeted gene, DKG50-P15VP4(BBa_K5531002), synthesized by a biotech company, contains proline-free heterotrimeric collagen-like protein motif DKG50 fused with the VP4 protein from the rotavirus with serotype P15. The pET24a(BBa_K5531005) serves as the vector. The homologous recombination method was employed for the construction of pET24a-DKG50-P15VP4(BBa_K5531007).

Figure 12. Plasmid map of pET24a-DKG50-P15VP4

Build 3:

We isolated pET24a vector from bacterial solution primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis’s result consistently predicted the expected outcome (DKG50-P15VP4 is 2000 bp). We selected several individual colonies for sequencing. Figure 13 shows the success of pET24a-DKG50-P15VP4 construction.

Figure 13. The results of pET24a-DKG50-P15VP4

Test 3:

Later, the plasmid mounted with the gene of DKG50-P15VP4 was transferred to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli BL21, which can help express His-DKG50-P15VP4. After the colony PCR of E.coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression.

After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis. The target protein DKG50-P15VP4 has a size of 68.1 kDa.

Figure 14. The expression of DKG50-P15VP4 using E.coli BL21

Learn 3:

We should continue optimizing the recombinant protein technology pathway for the vaccine, exploring various forms of the VP4 protein to improve its protective efficacy and identify the vaccine antigen. Additionally, we will develop adjuvants and formulations, focusing on designing adjuvants to boost immune responses and creating suitable vaccine formulations.

Part 4: BBa_K5531009 (pET-24a-EPG50-P8VP4)

Design 4:

Using the backbone of pET-24a(BBa_K5531005), proline-free heterotrimeric collagen-like protein motif EPG50 is fused with serological P8-VP4 via chemical synthesis. The homologous recombination method was employed for the construction of pET-24a-EPG50-P8VP4(BBa_K5531009).

Figure 15. Plasmid map of pET-24a-EPG50-P8VP4

Build 4:

We isolated pET-24a vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis result displayed correctness toward the expected outcome (EPG50-P8VP4 is 2000 bp). We selected several individual colonies for sequencing. Figure 16 shows the success of pET24a-EPG50-P8VP4 construction.

Figure 16. The results of pET24a-EPG50-P8VP4

Test 4:

Later, the plasmid mounted with the gene of EPG50-P8VP4 will then be transferred to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli BL21, which can help express His-EPG50-P8VP4. After the colony PCR of E.coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression.

After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein EPG50-P8VP4 has a size of 67.5 kDa.

Figure 17. The expression of EPG50-P8VP4 using E.coli BL21.

Learn 4:

After protein cell lysis and purification, it is important to ensure the timely use and proper storage of the protein. Store the purified protein in suitable conditions to maintain its stability and activity for future experiments. Additionally, avoid repeated freeze-thaw cycles and consider aliquoting the protein for multiple usages to minimize degradation and maintain its integrity.

Part 5: BBa_K5531008 (pET24a-PKG50-P4VP4)

Design 5:

This targeted gene, PKG50-P4VP4(BBa_K5531003), synthesized by a biotech company and constructed by a biotech company, contains proline-free heterotrimeric collagen-like protein motifs PKG fused P15VP4 protein. The pET24a(BBa_K5531005) serves as the vector. The homologous recombination method was employed for the construction of pET24a-PKG50-P4VP4(BBa_K5531008).

Figure 18. Plasmid map of pET-24a-PKG50-P4VP4

Build 5:

We isolated pET-24a vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis’s result displayed consistency toward the expected outcome (PKG50-P4VP4 is 2000 bp); We selected several individual colonies for sequencing. Figure 19 shows the success of pET24a-PKG50-P4VP4 construction.

Figure 19. The results of pET24a-PKG50-P4VP4

Test 5:

Later, the plasmid pET-24a-PKG50-P4VP4 will then be transferred to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli BL21, which can help express His-PKG50-P4VP4. After the colony PCR of E.coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression.

After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein PKG50-P4VP4 has a size of 68.1 kDa.

Figure 20. The expression of PKG50-P4VP4 using E.coli BL21

Learn 5:

During protein purification, it is important to follow standard operating procedures for purification columns. Careful loading of the protein sample onto the column is essential to prevent the formation of air bubbles and to ensure uniform distribution within the resin.

Other Test:

The collagen of the C-D-E-VP4 complex (in a 1:1:1 ratio) was diluted to a concentration of 0.5 mg/mL and incubated at 37°C for 1 hour prior to native-PAGE and SEC analysis. As illustrated in Figure 21, the samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation. Based on the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 21. The peaks for the C-D-E-VP4 complex also ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.57 CV, 0.68 CV, 0.72 CV, and 0.77 CV, indicating a trimer at 0.57 CV and the other peaks corresponding to the monomers of C, D, and E. It confirms the reliability of our experimental design.

Figure 21. Native-PAGE analysis of oligomeric states of collagen c-d-e complex(left); Size exclusion chromatographic (SEC) analysis of oligomeric states of collagen c-d-e complex(right)