Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3′ end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.
The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal.
Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment.
The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.
As an essential technique to construct DNA-based experimental tools for expression in prokaryotic and eukaryotic cells, molecular cloning is a very important component of experimental biology. Molecular cloning involves four processes: the acquisition of insert DNA, ligation of the insert into the appropriate vector, transformation of the recombinant vector into Escherichia coli (E. coli), and identification of the positive clones1. So far, multiple methods have been adopted for joining DNA molecules by using restriction enzymes2,3 and PCR-mediated recombination4,5,6. Homologous recombination, known as seamless cloning technology, is the group of cloning methods, which allows sequence-independent and scarless insertion of one or more fragments of DNA into a vector. This technology includes sequence- and ligation-independent cloning (SLIC), Seamless Ligation Cloning Extract (SLiCE), In-Fusion, and Gibson Assembly. It employs an exonuclease to degrade one strand of the insert and a vector to generate cohesive ends, and either in vivo repair or in vitro recombination to covalently join the insert to the vector by forming phosphodiester bonds. The ability to join a single insert to a vector at any sequence without any scars is very appealing. Furthermore, the technology has the ability to join 5-10 fragments in a predetermined order without sequence restrictions.
As one of many recombinant DNA techniques, the Gibson Assembly technique, currently the most effective cloning method7,8, is a robust and elegant exonuclease-based method to assemble one or multiple linearized DNA fragments seamlessly. The Gibson Assembly reaction is performed under isothermal conditions using a mixture of three enzymes,namely, 5' exonuclease, high-fidelity polymerase, and a thermostable DNA ligase. Single-strand 3´ overhangs created by the 5'-3' exonuclease contribute to the annealing of fragments that share complementarity at one end. The high-fidelity polymerase effectively fills the gaps in the annealed single-strand regions by adding dNTPs, and the thermostable DNA ligase seals the nicks to form joint DNA molecules8. Hence, this technical method has been widely used for the construction of gene expression vectors.
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that leads to reproductive impairment and respiratory failure in pigs caused by PRRSV at any age9. The syndrome is manifested as fever, anorexia, pneumonia, lethargy, depression, and respiratory distress. Moreover, clinical signs, including red/blue discoloration of the ears, have been observed in some epidemics. As a member of the family arterivirus, PRRSV is widely transmitted to pork-producing countries by direct contact and exchange of fluids, including urine, colostrum, and saliva. Due to the spread of PRRSV in the United States, the total economic losses of the pork industry have been estimated to be approximately $664 million per year, based on the breeding scale of 5.8 million sows and 110 million pigs10,11. The Animal and Plant Health Inspection Service report shows that 49.8% of unvaccinated pigs show the presence of PRRSV in serum12 and low levels of PRRSV in infected pigs are excreted through saliva, nasal secretions, urine, and feces13. Multiple strategies have been implemented to control PRRSV propagation14,15,16. In addition to elimination procedures to create completely virus-negative populations or improving biosafety and management, administering vaccines is an effective means of controlling PRRS.
PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a length of approximately 15 kilobases (kb). The PRRSV genome consists of at least 10 open reading frames (ORFs), a short 5' untranslated region (5' UTR), and a poly(A) tail at the 3' terminus (Figure 1A)17. The genome of a negative-stranded RNA virus is non-infectious whereas the genome from positive-stranded RNA viruses is infectious. There are two main strategies for RNA and DNA transfection for generating virus progeny18. However, cloning the full-length fragment corresponding to the RNA genome is crucial for the construction of infectious clones. Due to the long and complex nature of the PRRSV genome, the full-length genome cannot be easily obtained through PCR at once. Additionally, although the artificial synthesis of PRRSV genes is an effective solution, the synthesis of long fragments is often expensive. Hence, to obtain the PRRSV full-length expression vector, we attempted to create it by the multiple inserts homologous recombination method19,20. Unfortunately, we were not able to obtain the full-length gene expression vector. Therefore, in this study, we added appropriate restriction sites to the reverse primer and successfully obtained the pVAX1-PRRSV expression vector by several rounds of homologous recombination reactions. Furthermore, this method can also achieve deletion or mutation of target genes and efficiently join a large number of DNA fragments to the expression vector.
1. Preparation of the template of the PRRSV gene
2. PCR primer design
3. PCR to amplify fragments
4. Purification of the PCR fragments
NOTE: Purifying the PCR products from a gel using a gel extraction kit (see Table of Materials) is important for vector construction.
5. Preparation of a linearized vector
NOTE: After preparing the plasmid, the selected enzymes can be used to cut it. Long digestion or dual enzyme digestion is crucial for ensuring the digestion of all DNA. This will reduce the number of false-positive clones in subsequent experiments.
6. Subcloning to a new vector
NOTE: Good cloning efficiency can be achieved when using 50-200 ng of vector and inserts.
7. Analyzing the transformants
In this paper, we present an in vitro recombination system to assemble and repair overlapping DNA molecules using the reverse primer via continually introduced restriction sites (Figure 1B). This system is a simple and efficient procedure comprising the preparation of the linear vector and the insert fragments containing overhangs introduced by PCR with primers having appropriate 5' extension sequences and restriction sites; an in vitro single isothermal reaction and the standard chemical transformation of recombination products into suitable host bacteria; and the selection of a positive colony and obtaining the recombinant vector for the next round of cloning. For obtaining the pVAX1-PRRSV vector step by step, restriction sites must be absent from the inserts and multiple cloning sites of the recombinant vector.
As it is difficult to obtain full-length and complex gene sequences with a single PCR, insert fragments of PRRSV could only be amplified with six PCR reactions (Figure 1C). During the first round of recombination, Fragment 1 of PRRSV was successfully inserted into the pVAX1 vector, and the new recombinant plasmid with the introduced restriction sites (NheI, a single enzyme cleavage site) was named pVAX1-F1 (Figure 1D). At the second round of recombination, the pVAX1-F1 vector was linearized with the restriction enzyme NheI. Fragment 2 was successfully inserted into the pVAX1-F1 vector, yielding the recombinant plasmid pVAX1-F2 with restriction sites (NheI, a single enzyme cleavage site) (Figure 1D). For the third round of recombination, as shown in Figure 1D, fragment 3 was successfully inserted into the pVAX1-F2 vector to obtain pVAX1-F3 with restriction sites (NheI, a single enzyme cleavage site). In the fourth round of recombination, we obtained the pVAX1-F4 vector by introducing fragment 4 with restriction sites EcoRV (a single enzyme cleavage site). Then, the pVAX1-F4 vector was linearized by the restriction enzyme EcoRV and NotI. Fragment 5 was cloned into the pVAX1-F4 vector to obtain pVAX1-F5 with restriction sites (NotI, a single enzyme cleavage site). After the last round of recombination, the PRRSV full-length overexpression vector (pVAX1-PRRSV) was successfully obtained (Supplemental File 1). As illustrated in Figure 1D, the new recombinant plasmid becomes larger and larger with the continuous introduction of fragments. Thus, these results indicated that the combination of adding appropriate restriction sites and utilizing the Gibson Assembly technique can achieve the efficient introduction of DNA fragments into the vector.
Figure 1: Organization and construction of pVAX1-F6 (pVAX1-PRRSV) vectors by homologous recombination. (A) Organization of the PRRSV genome. ORF1b encodes RNA-dependent RNA polymerase, RNA helicase, and multinuclear zinc-binding domains. Other ORFs encode membrane-associated glycoproteins (GP2, GP3, GP4, and GP5), E protein, membrane envelope protein, and the nucleocapsid protein. (B) Schematic diagram of the process of generation of the pVAX1-PRRSV vector.(C) DNA agarose gel shows PCR amplification of target fragments. (D) With the insertion of fragments, the size of the recombinant vector increases gradually. Abbreviations: PRRSV = Porcine Reproductive and Respiratory Syndrome Virus; ORF = open reading frame; GP = glycoprotein; E = E protein; M = membrane envelope protein; N = nucleocapsid protein. Please click here to view a larger version of this figure.
Component | Volume | |
PRRSV RNA | 2 μL (25 ng) | |
RNase-free water | 11 μL | |
5x Reaction Mix | 4 μL | |
Supreme Enzyme Mix | 3 μL | |
Temperature | Duration | Cycles |
25 °C | 10 min | Remove genomic DNA, random primers pair with RNA templates |
55 °C | 15 min | Rapid inactivation of dsDNase and reverse transcription |
85 °C | 5 min | Reverse transcriptase inactivation |
Table 1: Reverse transcription setup and conditions.
Component | Volume | ||
5x SuperFi II Buffer | 10 μL | ||
10 μM Forward Primer | 2.5 μL | ||
10 μM Reverse Primer | 2.5 μL | ||
10 mM dNTPs | 1 μL | ||
cDNA (PRRSV) | 1 μL | ||
Platinum SuperFi II DNA Polymerase | 1 μL | ||
ddH2O | 32 μL | ||
Cycle step | Temperature | Duration | Cycles |
Initial denaturation | 98 °C | 30 s | 1 |
Denaturation | 98 °C | 10 s | 35 |
Annealing | 60 °C | 10 s | |
Extension | 72 °C | 3 min | |
Final extension | 72 °C | 5 min | 1 |
Hold | 4 °C | – | – |
Table 2: PCR reaction setup and conditions.
pVAX1 linearization | pVAX1-F1 linearization | pVAX1-F2 linearization | pVAX1-F3 linearization | pVAX1-F4 linearization | pVAX1-F5 linearization | |
Component | Volume | Volume | Volume | Volume | Volume | |
Vector | 10 μL (1 μg) of pVAX1 | 6 μL (1 μg) of pVAX1-F1 | 3.5 μL (1 μg) of pVAX1-F2 | 7.5 μL (1 μg) of of pVAX1-F3 | 10 μL (1 μg) of pVAX1-F4 | 15 μL (1 μg) of pVAX1-F5 |
Restriction enzyme 1 | 1 μL of NdeI | 1 μL of NdeI | 1 μL of NdeI | 1 μL of NdeI | 1 μL of EcoRV | 1 μL of NotI |
Restriction enzyme 2 | 1 μL of HindIII | 1 μL of HindIII | 0.5 μL of CIAP | 0.5 μL of CIAP | 1 μL of NotI | – |
10x FastDigest buffer | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL |
ddH2O | 6 μL | 10 μL | 13 μL | 9 μL | 6 μL | 2 μL |
Total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
Table 3: Linearization mixture of pVAX1, pVAX1-F1, pVAX1-F2, pVAX1-F3, pVAX1-F4, and pVAX1-F5.
Component | Volume |
ExonArt Seamless Cloning and Assembly kit | 5 μL |
Linearized Vector | 200 ng |
Fragment | 200 ng |
ddH2O | to 10 μL |
Table 4: ExonArt seamless cloning and assembly reaction setup.
Component | Volume | |
2x Universal Green PCR Master Mix | 10 μL | |
10 μM Forward Primer | 0.4 μL | |
10 μM Reverse Primer | 0.4 μL | |
Template | 1 μL | |
ddH2O | 8.2 μL | |
Total | 20 μL | |
Temperature | Duration | Cycles |
95 °C | 30 s | 1 |
98 °C | 10 s | 28 |
56 °C | 10 s | |
72 °C | 3 min | |
72 °C | 5 min | 1 |
4 °C | – | – |
Table 5: Colony PCR setup and conditions.
Supplemental Figure S1: Schematic of the process of primer design. Other primer designs such as P5 and P6 are also shown. Please click here to download this File.
Supplemental Figure S2: Primer sequences. Please click here to download this File.
Supplemental File 1: DNA sequencing data. Please click here to download this File.
The Gibson assembly technique is an in vitro recombination-based molecular cloning method for the assembly of DNA fragments8. This method enables the assembly of multiple DNA fragments into a circular plasmid in a single-tube isothermal reaction. However, one of the obstacles to the Gibson Assembly technique is the acquisition of long fragments from cDNA. The long fragments are difficult to accurately amplify for many reasons. For example, primers are easier to mismatch during long extending times, cDNA may not be of good quality, or GC-rich regions will stop DNA polymerizing. When the full-length fragment cannot be easily obtained from cDNA using PCR, the full-length fragment needs to be divided into multiple fragments for amplification. However, as the number and length of the inserted DNA fragments increase, the efficiency and positive clones from the homologous recombination assay will also significantly decrease. Hence, we tried an alternative solution by introducing suitable restriction sites at the 3' end of the inserts. Then, we could linearize the vector and join the DNA fragments into the vector one by one through homologous recombination technology and eliminate the unwanted restriction sites via the homologous recombination reaction.
As one of the most economically significant pathogens in the global markets, PRRSV has always attracted the attention of researchers during the past 30 years10,21,22. It is a positive-stranded RNA virus, approximately 15 kb in length, and the long DNA fragment cannot be easily amplified from cDNA. Hence, to investigate whether this method can be used for the cloning of long fragments, we employed the PRRSV as a template for full-length genome cloning. In this study, we divided the full-length sequence into six fragments and added appropriate restriction sites to the reverse primer, obtaining six fragments by PCR. We then successfully obtained the full-length pVAX1-PRRSV expression vector by several rounds of recombination reactions. In addition, the constructed PRRSV expression vector can serve as a template for in vitro transcription to obtain capped RNAs for transfection or mRNA vaccine research. Meanwhile, the constructed mRNA virus plasmid can be used for batch screening of antiviral natural compounds in vitro through molecular interaction techniques, such as surface plasmon resonance23,24. The pVAX1 plasmid meets the requirements because an in vitro transcription assay requires a high-copy plasmid with a T7 promoter. Besides, the pVAX1-PRRSV plasmid containing the CMV promoter can be transfected into BHK-21cells for viral genome expression25,26.
In summary, with this protocol, we demonstrate that long full-length genes can be divided into several fragments obtained by PCR amplification. It is important that the reverse primers are introduced into the appropriate restriction sites so that the full-length gene expression vector can be obtained by several rounds of recombination reactions. This method can be used for full-length genes that cannot be directly obtained by a single PCR or for full-length genes that cannot be obtained by homologous recombination of multiple inserts. Hence, this approach is an important supplement to the Gibson Assembly technique, we anticipate that it can be widely used for the cloning of long DNA fragments and fusion gene construction.
The authors have nothing to disclose.
This work was supported by the financial support of the doctoral research initiation funds provided by the China West Normal University (No. 20E059).
1 kb plus DNA Ladder | Tiangen Biochemical Technology (Beijing) Co., Ltd | MD113-02 | |
2x Universal Green PCR Master Mix | Rong Wei Gene Biotechnology Co., Ltd | A303-1 | |
Agarose | Sangon Biotech (Shanghai) Co., Ltd. | 9012-36-6 | |
Benchtop Microcentrifuge | Thermo Fisher Scientific Co., Ltd | FRESCO17 | |
Clean Bench | Sujing Antai Air Technology Co., Ltd | VD-650-U | |
DNA Electrophoresis Equipment | Cleaver Scientific Co., Ltd | 170905117 | |
DNA Loading Buffer (6x) | Biosharp Biotechnology Co., Ltd | BL532A | |
E. Z. N. A. Gel Extraction kit | Omega Bio-Tek Co., Ltd | D2500-01 | |
E.Z.N.A. Plasmid DNA Mini Kit I | Omega Bio-Tek Co., Ltd | D6943-01 | |
Electro-heating Standing-temperature Cultivator | Shanghai Hengyi Scientific Instrument Co., Ltd | DHP-9082 | |
ExonArt Seamless Cloning and Assembly kit | Rong Wei Gene Biotechnology Co., Ltd | A101-02 | |
ExonScript RT SuperMix with dsDNase | Rong Wei Gene Biotechnology Co., Ltd | A502-1 | |
FastDigest Eco321 (EcoRV) | Thermo Fisher Scientific Co., Ltd | FD0303 | |
FastDigest HindIII | Thermo Fisher Scientific Co., Ltd | FD0504 | |
FastDigest NheI | Thermo Fisher Scientific Co., Ltd | FD0974 | |
FastDigest NotI | Thermo Fisher Scientific Co., Ltd | FD0596 | |
Gel Doc XR | Bio-Rad Laboratories Co., Ltd | 721BR07925 | |
Goldview Nucleic Acid Gel Stain | Shanghai Yubo Biotechnology Co., Ltd | YB10201ES03 | |
Ice Maker Machine | Shanghai Bilang Instrument Manufacturing Co., Ltd | FMB100 | |
Invitrogen Platinum SuperFi II DNA Polymerase | Thermo Fisher Scientific Co., Ltd | 12361010 | |
LB Agar Plate (Kanamycin) | Sangon Biotech (Shanghai) Co., Ltd. | B530113-0010 | |
LB sterile liquid medium (Kanamycin) | Sangon Biotech (Shanghai) Co., Ltd. | B540113-0001 | |
Micropipettors | Thermo Fisher Scientific Co., Ltd | — | |
Microwave Oven | Panasonic Electric (China) Co., Ltd | NN-GM333W | |
Orbital Shakers | Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd | ZHWY-2102C | |
PRRSV virus | Sichuan Agricultural University | — | |
SnapGene | GSL Biotech, LLC | v5.1 | To design primers |
T100 PCR Gradient Thermal Cycler | Bio-Rad Laboratories Co., Ltd | T100 Thermal Cycler | |
TAE buffer | Sangon Biotech (Shanghai) Co., Ltd. | B040123-0010 | |
TRIzol Reagent | Thermo Fisher Scientific Co., Ltd | 15596026 | RNA extraction reagent |