cloning of the porcine reproductive and respiratory syndrome virus gene into the pvax1 vector

שיבוט הגן PRRSV באורך מלא בווקטור הביטוי pVAX1

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&#39; exonuclease, high-fidelity polymerase, and a thermostable DNA ligase. Single-strand 3&#180; overhangs created by the 5&#39;-3&#39; 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&#39; untranslated region (5&#39; UTR), and a poly(A) tail at the 3&#39; 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.

מחבר זרקור: חקר טכניקות שיבוט עבור מקטעי DNA באורך מלא

גישת שיבוט חלקה לבנייה וקטורית של ביטוי וירוסים ברבייה חזירית ובתסמונת הנשימה

This protocol demonstrates the construction of a lengthy and intricate gene expression vector. When a full-length DNA fragment cannot be obtained by a single PCR from cDNA, or the full-length gene expression vector cannot be obtained by multiple-insert homologous recombination in vitro, this method is applicable. Currently, multiple-insert ligation cloning is used to obtain full-length DNA fragment for cloning into a suitable vector.This method can achieve deletion or mutation of target genes and efficiently join a large number of DNA fragments to the expression vector.

שיבוט הגן של נגיף הרבייה והנשימה החזירית לווקטור pVAX1

התחל על ידי ליניאריזציה של הווקטור. הכינו את תערובת התגובה המכילה אנזימי הגבלה ספציפיים בטמפרטורת החדר. השתמשו בערכת מיצוי ג'ל כדי לטהר את הווקטורים הלינאריים, ולאחר מכן דגרו על 37 מעלות צלזיוס למשך 60 דקות.לאחר מכן, הרכיבו את תגובת השיבוט וההרכבה החלקה של exon R כדי לשכפל את הגן PRRSV. התאם את נפח התגובה הכולל ל -10 מיקרוליטר עם מים מעוקרים שעברו דה-יוניזציה. לאחר מכן, מערבבים היטב.לדגור את התערובת thermocycler במשך 15 עד 60 דקות ב 50 מעלות צלזיוס. לאחר מכן, אחסנו את הדגימות על קרח. לאחר הפיכת הווקטור לתאים מוכשרים, בחרו שמונה מושבות ל-20 מיקרוליטר של מדיום LB המכיל קנמיצין.הגדר תגובת PCR של מושבה כדי לנתח את הטרנספורמטים. וקטור ביטוי יתר PRRSV באורך מלא התקבל על ידי החדרת מקטע PRRSV אחד לתוך וקטור pVAX1, יצירת pVAX1-F1. סבבים רצופים של רקומבינציה תוך שימוש באנזימי הגבלה ספציפיים הביאו לשילוב של מקטעים שניים עד חמישה, שהודמיינו על ידי עלייה בגודל הפלסמיד.

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Research

JoVE Journal

Biology

Cloning of the Porcine Reproductive and Respiratory Syndrome Virus Gene into the pVAX1 Vector

27 Views. התחל על ידי ליניאריזציה של הווקטור. הכינו את תערובת התגובה המכילה אנזימי הגבלה ספציפיים בטמפרטורת החדר. השתמשו בערכת מיצוי ג'ל כדי לטהר את הווקטורים הלינאריים, ולאחר מכן דגרו על 37 מעלות צלזיוס למשך 60 דקות.לאחר מכן, הרכיבו את תגובת השיבוט וההרכבה החלקה של exon R כדי לשכפל את...

Video: שיבוט הגן של נגיף הרבייה והנשימה החזירית לווקטור pVAX1

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

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&#39; end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3&#39; 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.

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 ...

PCR Primer Design for Cloning of the Porcine Reproductive and Respiratory Syndrome Virus Gene

To begin, open the software and select the option to create a new DNA file. Prepare the PRRSV gene sequence from NCBI and click Okay to generate the sequence files. Analyze the sequence and mark the fragment junctions.Identify all the junctions before designing the specific sequence primers for the fragments. Then click on Primers and choose Add Primer. Paste the specific sequence and add overlap sequences of the vector to the first five prime nucleotide of the primer.Name the forward primer and click on Add Primer to Template to incorporate the designed primer into the template. Then mark the fragment junctions and design the reverse specific sequence primer of the fragments. Again, navigate to Primers and select Add Primer.Input the specific sequence. Add the restriction site to the first five prime nucleotide. Also, include 20 to 40 base pairs of vector overhang sequences to the first five prime nucleotide of the restriction site.Name the reverse primer and select Add Primer to Template. Next, set up six individual PCR reactions using the six fragments and designed primer pairs. Run the PCR following a three-step protocol as shown here.After PCR completion, pipette one microliter of six xDNA loading buffer with five microliters of the PCR product. Mix and centrifuge briefly. Analyze the samples using 1%agarose gel electrophoresis.To construct a full length gene, insert fragments of PRRSV were amplified using six PCR reactions. The sizes of the fragments were confirmed by agarose gel electrophoresis.

תכנון פריימר PCR לשיבוט הגן של נגיף הרבייה והנשימה החזירית

Begin by linearizing the vector. Prepare the reaction mixture containing specific restriction enzymes at room temperature. Use a gel extraction kit to purify the linearized vectors, then incubate a 37 degrees Celsius for 60 minutes.Then, assemble the exon R seamless cloning and assembly reaction to sub-clone the PRRSV gene. Adjust the total reaction volume to 10 microliters with sterilized deionized water. Then, thoroughly mix.Incubate the mixture in a thermocycler for 15 to 60 minutes at 50 degrees Celsius. Afterwards, store the samples on ice. After transforming the vector into competent cells, pick eight colonies into 20 microliters of LB medium containing kanamycin.Set up a colony PCR reaction to analyze the transformants. A full length PRRSV over-expression vector was obtained by introducing PRRSV fragment one into the pVAX1 vector, creating pVAX1-F1. Successive rounds of recombination using specific restriction enzymes resulted in incorporation of fragments two through five, visualized by an increase in plasmid size.