A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction

Summary

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.

Abstract

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.

Introduction

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 enzymes^2,3 and PCR-mediated recombination^4,5,6. 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 method^7,8, 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 pigs^10,11. 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 propagation^14,15,16. 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 method^19,20. 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.

Protocol

1. Preparation of the template of the PRRSV gene

1. Thaw the virus stock in 1 mL of RNA extraction reagent (see Table of Materials).
2. Add 0.2 mL of chloroform and mix thoroughly. Incubate for 3 min.
3. Centrifuge the mixture for 15 min at 12,000 × g at 4 °C.
   NOTE: The mixture is divided into three phases, namely, a colorless aqueous phase, an interphase, and a red phenol-chloroform phase.
4. Pipet the colorless aqueous phase out and transfer it to a new tube.
5. Mix the aqueous phase thoroughly with 0.5 mL of isopropanol and incubate for 10 min at 4 °C.
6. Centrifuge for 10 min at 12,000 × g at 4 °C.
   NOTE: The bottom of the tube has a white RNA precipitate.
7. Use a micropipette to discard the supernatant of the tube.
8. Add 1 mL of 75% ethanol to resuspend the pellet and vortex briefly.
9. Centrifuge for 5 min at 7,500 × g at 4 °C. Use a micropipette to discard the supernatant of the tube.
10. Dry the RNA for 5 min, add 20-50 µL of RNase-free water to resuspend the RNA, and mix thoroughly.
11. Proceed to perform reverse transcription; set up the reactions to perform reverse transcription as shown in Table 1.
    NOTE: To ensure successful reverse transcription, use high-quality RNA templates.
12. Use the resulting cDNA for PCR or store it at -20 °C.

2. PCR primer design

1. Designing the forward primer
   1. Open the software and choose New DNA File.
   2. Paste the PRRSV gene sequence (GenBank: FJ548852.1) from NCBI into the software. Click OK to generate the sequence files.
   3. Analyze the sequence and mark the fragment junctions. Design the forward specific sequence primer of the fragments. For most cases, the preferable melting temperature (Tm) is between 55 °C and 62 °C, and the GC content is 40-60%.
   4. Click on Primers and choose Add Primer.
   5. Paste the specific sequence and add overlap sequences of the vector to the first 5' nucleotide of the specific sequence primer. Near each junction, choose 20-40 bp to serve as the overlap region between the two adjacent fragments.
   6. Name the forward primer containing the overhangs and the specific sequence (see Supplemental Figure S1).
   7. Click on Add Primer to Template.
2. Designing the reverse primer
   1. Analyze the sequence and mark the fragment junctions in the software. Design the reverse specific sequence primer of the fragments.
   2. Click on Primers and choose Add Primer.
   3. Paste the specific sequence and add the restriction site to the first 5' nucleotide of the specific sequence primer.
      NOTE: The restriction sites added must be absent from the insert fragment and the vector except for the multiple cloning sites.
   4. Add 20-40 bp overhang sequences of vectors to the first 5' nucleotide of the restriction site.
   5. Name the reverse primer containing the overhangs and the specific sequence (see Supplemental Figure S1).
   6. Click on Add Primer to Template.

3. PCR to amplify fragments

1. Set up six individual PCR reactions (Table 2): for fragment 1, use primers P1 and P2 (see Supplemental Figure S2); for fragment 2, use primers P3 and P4 (see Supplemental Figure S2); for fragment 3, use primers P5 and P6 (see Supplemental Figure S2); for fragment 4, use primers P7 and P8 (see Supplemental Figure S2); for fragment 5, use primers P9 and P10; and use primers P11 and P12 to amplify fragment 6 (see Supplemental Figure S2).
   NOTE: Thaw, mix, and briefly centrifuge each component before use.
2. Run the PCR using the three-step protocol in Table 2.
3. Add 1 µL of 6x DNA loading buffer to 5 µL of PCR product, mix, and briefly centrifuge the contents.
4. Analyze the samples using 1% agarose gel electrophoresis.

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.

1. Add 9 µL of 6x DNA loading buffer to 45 µL of PCR products, mix, and briefly centrifuge the contents.
2. Perform 1% agarose gel/goldview electrophoresis to separate the DNA fragments.
   NOTE: Do not reuse TAE running buffer as its pH value will affect DNA fragment recovery.
3. Upon adequate separation of bands, use a sharp scalpel to carefully excise the DNA bands.
   NOTE: Minimize the size of the gel slice by trimming off excess agarose.
4. Weigh the gel slice in a clean 1.5 mL microcentrifuge tube, add an equal volume of binding buffer to the gel slice (e.g., 0.3 mL to a 0.3 g slice), and incubate at 60 °C for 7 min.
5. Insert a mini column in a 2 mL collection tube. Add 700 µL of DNA/agarose solution from step 4.4 to the mini column.
6. Centrifuge at 10,000 × g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.
7. Add 300 µL of binding buffer and centrifuge at 13,000 × g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.
8. Add 700 µL of wash buffer and centrifuge at 13,000 × g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.
9. Spin the empty mini column for 2 min at maximum speed to dry the column matrix. Place the mini column in a clean 1.5 mL microcentrifuge tube.
10. Add 20 µL of deionized water directly to the center of the column membrane and let sit at room temperature for 2 min. Centrifuge at 13,000 × g speed for 1 min.
11. Store the DNA at -20 °C.

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.

1. pVAX1 linearization
   1. Prepare the reaction mixture at room temperature in the order indicated (Table 3).
      NOTE: The volume of water should be added to keep the indicated total reaction volume. Here, 1 µg of the plasmid was digested with enzymes in the reaction mixture. Depending on the plasmid concentration, the volume of the plasmid can be adjusted in the reaction mixture.
   2. Mix gently; then spin down. Incubate at 37 °C in a heat block or water thermostat for 60 min.
   3. Perform gel purification similar to the purification of the PCR fragments in section 4 using the gel extraction kit (see Table of Materials).
2. pVAX1-F1 linearization
   NOTE: pVAX1-F1 is the vector obtained from the first round of pVAX1 (NdeI and HindIII) and fragment 1 recombination. pVAX1-F2 is the vector obtained from the round of pVAX1-F1 (NdeI) and fragment 2 recombination. pVAX1-F3 is the vector obtained from the round of pVAX1-F2 (NdeI) and fragment 3 recombination. pVAX1-F4 is the vector obtained from the round of pVAX1-F3 (NdeI) and fragment 4 recombination. pVAX1-F5 is the vector obtained from the round of pVAX1-F4 (EcoRV and NtoI) and fragment 5 recombination.
   1. For each vector, prepare the reaction mixture separately at room temperature in the order indicated (Table 3).
   2. Mix gently; then spin down. Incubate at 37 °C in a heat block or water thermostat for 60 min.
   3. Perform gel purification similar to the purification of the PCR fragments in section 4 using the gel extraction kit (see Table of Materials).

6. Subcloning to a new vector

NOTE: Good cloning efficiency can be achieved when using 50-200 ng of vector and inserts.

1. Set up the ExonArt seamless cloning and assembly reaction (Table 4). Adjust the total reaction volume to 10 µL using sterilized deionized H₂O and mix.
2. Incubate the reaction in a thermocycler for 15-60 min at 50 °C. Store the samples on ice.
   NOTE: Extending the incubation up to 60 min may enhance assembly efficiency.
3. Thaw DH5α chemically competent cells on ice.
4. Add 10 µL of the assembly product to the competent cells; then, mix gently by pipetting up and down.
5. Place the mixture on ice for 30 min.
6. Heat shock at 45 °C for 45 s and transfer the tubes onto ice for 3 min.
7. Add 900 µL of SOC medium to the tube.
8. Shake the tube at 225 rpm for 1 h in a 37 °C shaking incubator.
9. Centrifuge the transformation reaction at 6,000 × g for 2 min. Discard the supernatant and resuspend the cells in 100 µL of fresh SOC medium.
10. Spread the transformed cell suspension on a separate LB plate with 50 µg/mL kanamycin.
11. Incubate all plates overnight at 37 °C. Pick individual isolated colonies from each experimental plate.

7. Analyzing the transformants

1. Pick eight colonies into 20 µL of LB medium containing 50 µg/mL kanamycin.
2. Set up the colony PCR reaction and run the PCR (Table 5).
   NOTE: For the colony PCR reaction for fragment 1, use primers P1 and P2 (see Supplemental File 1); for fragment 2, use primers P3 and P4 (see Supplemental File 1); for fragment 3, use primers P5 and P6 (see Supplemental File 1); for fragment 4, use primers P7 and P8 (see Supplemental Figure S2); for fragment 5, use primers P9 and P10; and use primers P11 and P12 to detect fragment 6 (see Supplemental Figure S2).
3. Add 1 µL of 6x DNA loading buffer to 5 µL of the PCR product, mix, and briefly centrifuge the contents.
4. Analyze the results using 1% agarose gel electrophoresis.
5. Select one positive colony into 5 mL of LB medium containing 50 µg/mL kanamycin and grow overnight at 37 °C.
6. Obtain the plasmid using a plasmid DNA mini kit (see Table of Materials) according to the manufacturer's instructions.
7. Visualize the results using 1% agarose gel electrophoresis.

Results

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 th...

Discussion

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 extend...

Materials

Name	Company	Catalog Number	Comments
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