BEE VENOM FOR BACULOVIRUS
What is Baculovirus?
We can describe a baculovirus as enveloped viruses, which infect the insect larvae mostly from Lepidoptera. If we look at their genetic structure, they are found to be circular in shape and double-stranded DNA molecules having the size of 80 to 180 kbp. These molecules are packed in the form of rod-shaped nucleocapsids having a length of 200-400nm and a diameter of 40-50nm.
There are different versions of baculovirus, but AcMNPV (Autographa californica multiple nucleopolyhedroviruses is the most common type; which is used in different clinical studies and utilization of biological processes.
How Does It Infect?
It infects in the same manner as other viruses but produces two different phenotypes during its infection cycle. ODV (Occlusion Derived Viruses) initiates infection right from the larvae midget. It is also known as the primary infection stage. After this, the viral progeny involves BV (Budded Viruses) that are responsible for carrying out the systematic infection.
Such types of virions differ in their mechanism of delivering an infection and that depends on their cell types; the ODV infects midgut epithelial cells up to 10,000 more than the BV. On the other hand, BV is 1000 times more efficient than ODV – when it comes to infecting cultured cells.
BV phenotype is generally responsible for mediating viral propagation in the cell culture. But most of the information available, related to the baculovirus infection mechanism, is based on different studies conducted on insect cells that are infected by BV.
Expression of a Bee Venom Phospholipase A2 from Apis Cerana Cerana in the Baculovirus-Insect CellAbstract
BVPLA2, also known as, bee venom phospholipase A2, is a liplolutic enzyme, which catalyzes the hydrolysis of the sn-2 acyl bond of the glycerophospholipids to liberate the lysophospholipids and fatty acids. In this clinical study, AccPLA2 obtained from Apis cerana cerana, a Chinese honeybee’s venom was injected in the bacmid to develop a recombinant transfer vector. The Tn-5B-4(Tn) cells were infected with recombinant bacmid DNA. According to SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis, it was discovered that the double band molecules weighted 16-18 kDA. And the final product of hexahistidine AccPLA2 fusion protein showed an accumulation of up to 5.3% of the available cellular proteins.
AccPLA2 fusion protein also showed a cross-reaction with the BVPLA2 of Apis mellifera (European honey bee) polyclonal serum. This reaction leads to the formation of a double glycosylation band that is in agreement with the band created by native AmPLA2 – according to the Western blot analysis. PLA2 activity of the extracted cellular protein is about 3.16µmol/ (min-mg) in a hydrolyzing egg yolk. In the end, the recombinant protein AccPLA2 can be used to glycosylate Tn cells. In light of these findings, the results obtained provide the basic knowledge required for potentially engineering genetics to produce AccPLA2 for pharmaceutical purposes.
Introduction
PLA2 and EC 3.1.1.4 is a huge family of lipolytic enzymes, which catalyze the hydrolysis of the sn-2 bond of glycerophospholipids to set free lysophospholipids and fatty acids. PLA2 has various essential functions when it comes to cellular processing; which regulates the discharge of arachidonic acid and acts as a precursor of the eicosanoids of a potent inflammatory mediator. Furthermore, PLA2 plays an integral part in delaying cell death due to the oxidant induction, membrane remodeling, signal transduction processes, and atherosclerosis and host defense. PLA2 is also linked with different human disorders like endotoxic and septic shock, myocardial infarction, respiratory distress syndrome, autoimmune uveitis, and rheumatoid arthritis.
The PLA2 enzyme can be found from different sources like synovial fluids, insect and reptile venom, and mammalian pancreas. These enzymes are classified into 15 different, but closely related, groups according to their level of homology-based on amino acid and nucleotide sequences. We can say that PLA2 can be divided into 3 different groups for clinical examination.
Group I consists of mammalian pancreases and krait venom, Group II consists of viper and crotalid venom and the last group, Group III, consists of scorpion/lizard/bee venom. BVPLA2 is a member of the Group III and nearly makes up 12% of the total dry weight of bee venom found in Apis millifera. We can say that BVPLA2 is the strongest honeybee venom peptide. Naturally, it behaves like an allergen that cooperates with linked components to defend a bee colony against any sort of intruders and predators.
Melittin is another constituent of bee venom, which accelerates the activity level of BVPLA2. BVPLA2 possesses several pharmacological traits that include neurite outgrowth induction, myotoxicity, and neurotoxicity and HIV activity. There is a fresh nucleotide sequence of the BVPLA2 found from Apis millifera. AmPLA2's amino acid sequences contain the proregion of PLA2 (peptide of 18 amino acid) residues. It is a mature peptide that contains a 10-cystine residue that can form 5-disulfide bonds. Catalyzing activity and crystal structure of PLA2A have been documented in different clinical studies, carried out a few years ago.
Apis cerana, also known as the Asiatic honeybee, is also of the most common species of honey bee found in the south and southeastern parts of Asia. It is thought that Apis cerana, subspecies of the Asiatic honeybee is more than 3000 years old. Chinese farmers are well aware of the importance of these honeybees and have successfully maintained a colony of 1.2 million Apis cerana. On the other hand, European honey bee, also known as Apis millifera has a huge colony of 7.8 million bees.
One of the most interesting things is that the venom of both, Chinese and European honey bees have their biological characters and properties. In a previous clinical study, the BVPLA2 gene was amplified, which encodes an advanced peptide of Chinese honeybee. It was found that the general expression of AccPLA2 surfaces from 5d to 42d of worker bees of the Chinese honeybee. This study was set up to examine the expression, enzymatic activity, immunologic properties and glycosylation of recombinant AccPLA2 in the insect cells.
Method
PGEM-AcPLA2 was developed according to the method prescribed while; the Tn-5B-4 (Tn) cell line was maintained carefully in the lab. New Zealand rabbits were obtained from the Chinese Traditional Medical Institute of Zhejiang. Restriction enzymes HindIII and EcoRI, DL1500 and DL2000 markers were obtained from the TaKaRa Company, China. Native AmPLA2, which was commercially purified, was obtained from the Sigma Company, USA. E. coli strains DH10B, transfer vector pFastBacHTa, bovine calf serum and TNM-FH insect cell, Lipofectin, (IPTG) isopropyl beta-D-thiogalactopyranoside, X-gal and T4 DNA ligase were obtained from the Invitrogen Corporation, USA. Goat anti-rabbit IgG conjugate, NC filter, protein molecular marker, and Taq DNA polymerase were obtained from Sino Promega Company, China. While the plasmid DNA extraction kit was obtained from Omega Bio Tek Corporation, USA. Bovine serum albumin, sodium deoxycholate, and calcium chloride were obtained from the Sangon Company, China. It was made sure that all of the biochemical reagents, obtained from different sources, were of the highest commercially available level of purification.
The New Zealand rabbits were given a mixture of 300mg purified AmPLA2 along with 1ml of PBS. These rabbits were subjected to bleeding for over 3 weeks after their primary immunization was done through 200mg of the enzyme, which was emulsified in an incomplete PBS (1ml). After that, an injection having 200mg of enzyme dissolved in 1ml of saline was administered for a continuous period of 3 weeks. A blood sample obtained was analyzed to detect the effect value of the serum given. It was done through native AmPLA2 after 1 week of the second dose of enhanced immunization, managed through gel diffused protocol as put forward by the Sambrook and Russell. Serum obtained was stored at a very low temperature of -80 degrees centigrade.
Intracellular proteins of infected cells, native AmPLA2, and normal insect cells were examined through 10% (v/v) SDS-PAGE, again according to the method described by Sambrook and Russell. The gels were stained with the Coomassie brilliant blue R250. Expressed fusion protein's concentration was evaluated through thin layer scanning of the SDS-PAGE gel. Unstained protein, separated, was transferred to unmodified NC membrane for Western blots. This process was carried out in a special Bio-Rad transblot set up. The fusion protein was examined with the help of a rabbit antiserum against the native AmPLA2. Proteins were detected on the blot through goat anti-rabbit IgG conjugate as prescribed by Sambrook and Russell.
PLA2's titrimetric assay was carried out by a revised method, previously used by Owen et al. Fresh egg yolk substrate, which was blended in 200ml of deionized and distilled water, was made fresh on daily basis. The egg mixture was subjected to a heat of 37 degrees centigrade and later NaOH was mixed in to maintain its pH value at 8.0. This process was done in a special thermostatted micro reaction vessel. PH stat titration was carried out through the pH meter system, which helped in keeping pH value at 8.0 after adding 0.005mol/L of NaOH. Solubilized protein extracts were measured carefully for their concentration of infected cells and later they were added to substrate mixture. Purified AmPLA2 and BV powder, which was diluted in deionized and distilled water were used a positive control while extracts of Tn cells were chosen for the negative control.
Result
EcoRI/Hind digested fragment of AccPLA2 (cDNA) from pGem- AccPLA2 was administered into transfer vector pFastBacHTa. Products of the registered digestion and the PCR of recombinant plasmid had a 400bp band – quite identical to AccPLA2 fragment. The nucleotide sequencing also confirmed that inserted fragment gives a correct ORF for protein expression. According to the schematic presentation of the recombinant transfer vector, the inserted gene was found to be under control of autographa californica nuclear polyhedrosis virus polyhedrin promoter. Expressed gene was naturally designed to establish a fusion protein that involves hexahistidine resides at the amino terminus that eventually allows single-step purification with immobilized Ni2+ ion affinity chromatography.
Once recombinant transfer vector pFastBacHTa – AccPLA2 was converted into E.coli DH10B capable cells, which contain the baculovirus shuttle vector bacmid, it was further cultured. PCR identification was then conducted with bacmid DNA that was isolated from positive colonies in the form of templates making use of primer PUC/M13 and AccPLA2, respectively. 2 fragments, one about 2700bp and the other 100bp were found. After examination, they were found to be like the AccPLA2 fragment within MCS (multiple cloning sites). On the other hand, a bacmid fragment of 2300bp was located within the left and right range of the bacterial transposon TN7 + AccPLA2 fragment. It confirms that rBacmid-AccPLA2 and recombinant bacmid had been accurately developed.
With the help of Lipofection, rBacmid-AccPLA2 DNA was made available in the Tn cells in medium. AccPLA2 showed in 3d after the infection of rBacmid-AccPLA2 DNA. Once the centrifugation was completed for generating cells, amplified rBacmid in the Tn cells, rTnV-Bac-AccPLA2, was used in supernatants for propagation. Once propagation was completed for the 4 generations, rTnV-Bac-AccPLA2 genomic DNA was used for performing PCR identification from infected cells. Agarose electrophoretic analysis depicted that PCR products had 400bp, which was identical to the AccPLA2 gene; it was hence confirmed that rTnV-Bac-AccPLA2 genomic DNA had been successfully propagated in the Tn cells.
Discussion
Before this clinical study, BVPLA2 had not been expressed in any insect cells. But here the AccPLA2 gene was expressed successfully in the Tn cells. It was established that the expressed fusion protein was found to be active, indicating that the Tn cells can express a properly modified AccPLA2. The findings of this clinical study are totally in harmony with the results of previous clinical reports - Soldatova et al, Altmann et al and Li et al. Glycosylation of the recombinant protein can be termed as an important mechanism that directly affects protein function. This study, in combination with the previous studies, can provide a scientific base for the molecular biological application of AccPLA2 in the future.
Conclusion
Tn cells were constructed by transfecting recombinant bacmid DNA, which was made a part of a new BVPLA2 gene through apis cerana cerana glands to help express recombinant AccPLA2 protein. It is a BVPLA2 type structure that has nearly the same level of biological activity; that is it can be easily glycosylated in the Tn cells. This clinical study can provide basic, but helpful information essential for engineering genetics that can be used for producing AccPLA2 in the pharmaceutical industry.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865836/
We can describe a baculovirus as enveloped viruses, which infect the insect larvae mostly from Lepidoptera. If we look at their genetic structure, they are found to be circular in shape and double-stranded DNA molecules having the size of 80 to 180 kbp. These molecules are packed in the form of rod-shaped nucleocapsids having a length of 200-400nm and a diameter of 40-50nm.
There are different versions of baculovirus, but AcMNPV (Autographa californica multiple nucleopolyhedroviruses is the most common type; which is used in different clinical studies and utilization of biological processes.
How Does It Infect?
It infects in the same manner as other viruses but produces two different phenotypes during its infection cycle. ODV (Occlusion Derived Viruses) initiates infection right from the larvae midget. It is also known as the primary infection stage. After this, the viral progeny involves BV (Budded Viruses) that are responsible for carrying out the systematic infection.
Such types of virions differ in their mechanism of delivering an infection and that depends on their cell types; the ODV infects midgut epithelial cells up to 10,000 more than the BV. On the other hand, BV is 1000 times more efficient than ODV – when it comes to infecting cultured cells.
BV phenotype is generally responsible for mediating viral propagation in the cell culture. But most of the information available, related to the baculovirus infection mechanism, is based on different studies conducted on insect cells that are infected by BV.
Expression of a Bee Venom Phospholipase A2 from Apis Cerana Cerana in the Baculovirus-Insect CellAbstract
BVPLA2, also known as, bee venom phospholipase A2, is a liplolutic enzyme, which catalyzes the hydrolysis of the sn-2 acyl bond of the glycerophospholipids to liberate the lysophospholipids and fatty acids. In this clinical study, AccPLA2 obtained from Apis cerana cerana, a Chinese honeybee’s venom was injected in the bacmid to develop a recombinant transfer vector. The Tn-5B-4(Tn) cells were infected with recombinant bacmid DNA. According to SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis, it was discovered that the double band molecules weighted 16-18 kDA. And the final product of hexahistidine AccPLA2 fusion protein showed an accumulation of up to 5.3% of the available cellular proteins.
AccPLA2 fusion protein also showed a cross-reaction with the BVPLA2 of Apis mellifera (European honey bee) polyclonal serum. This reaction leads to the formation of a double glycosylation band that is in agreement with the band created by native AmPLA2 – according to the Western blot analysis. PLA2 activity of the extracted cellular protein is about 3.16µmol/ (min-mg) in a hydrolyzing egg yolk. In the end, the recombinant protein AccPLA2 can be used to glycosylate Tn cells. In light of these findings, the results obtained provide the basic knowledge required for potentially engineering genetics to produce AccPLA2 for pharmaceutical purposes.
Introduction
PLA2 and EC 3.1.1.4 is a huge family of lipolytic enzymes, which catalyze the hydrolysis of the sn-2 bond of glycerophospholipids to set free lysophospholipids and fatty acids. PLA2 has various essential functions when it comes to cellular processing; which regulates the discharge of arachidonic acid and acts as a precursor of the eicosanoids of a potent inflammatory mediator. Furthermore, PLA2 plays an integral part in delaying cell death due to the oxidant induction, membrane remodeling, signal transduction processes, and atherosclerosis and host defense. PLA2 is also linked with different human disorders like endotoxic and septic shock, myocardial infarction, respiratory distress syndrome, autoimmune uveitis, and rheumatoid arthritis.
The PLA2 enzyme can be found from different sources like synovial fluids, insect and reptile venom, and mammalian pancreas. These enzymes are classified into 15 different, but closely related, groups according to their level of homology-based on amino acid and nucleotide sequences. We can say that PLA2 can be divided into 3 different groups for clinical examination.
Group I consists of mammalian pancreases and krait venom, Group II consists of viper and crotalid venom and the last group, Group III, consists of scorpion/lizard/bee venom. BVPLA2 is a member of the Group III and nearly makes up 12% of the total dry weight of bee venom found in Apis millifera. We can say that BVPLA2 is the strongest honeybee venom peptide. Naturally, it behaves like an allergen that cooperates with linked components to defend a bee colony against any sort of intruders and predators.
Melittin is another constituent of bee venom, which accelerates the activity level of BVPLA2. BVPLA2 possesses several pharmacological traits that include neurite outgrowth induction, myotoxicity, and neurotoxicity and HIV activity. There is a fresh nucleotide sequence of the BVPLA2 found from Apis millifera. AmPLA2's amino acid sequences contain the proregion of PLA2 (peptide of 18 amino acid) residues. It is a mature peptide that contains a 10-cystine residue that can form 5-disulfide bonds. Catalyzing activity and crystal structure of PLA2A have been documented in different clinical studies, carried out a few years ago.
Apis cerana, also known as the Asiatic honeybee, is also of the most common species of honey bee found in the south and southeastern parts of Asia. It is thought that Apis cerana, subspecies of the Asiatic honeybee is more than 3000 years old. Chinese farmers are well aware of the importance of these honeybees and have successfully maintained a colony of 1.2 million Apis cerana. On the other hand, European honey bee, also known as Apis millifera has a huge colony of 7.8 million bees.
One of the most interesting things is that the venom of both, Chinese and European honey bees have their biological characters and properties. In a previous clinical study, the BVPLA2 gene was amplified, which encodes an advanced peptide of Chinese honeybee. It was found that the general expression of AccPLA2 surfaces from 5d to 42d of worker bees of the Chinese honeybee. This study was set up to examine the expression, enzymatic activity, immunologic properties and glycosylation of recombinant AccPLA2 in the insect cells.
Method
PGEM-AcPLA2 was developed according to the method prescribed while; the Tn-5B-4 (Tn) cell line was maintained carefully in the lab. New Zealand rabbits were obtained from the Chinese Traditional Medical Institute of Zhejiang. Restriction enzymes HindIII and EcoRI, DL1500 and DL2000 markers were obtained from the TaKaRa Company, China. Native AmPLA2, which was commercially purified, was obtained from the Sigma Company, USA. E. coli strains DH10B, transfer vector pFastBacHTa, bovine calf serum and TNM-FH insect cell, Lipofectin, (IPTG) isopropyl beta-D-thiogalactopyranoside, X-gal and T4 DNA ligase were obtained from the Invitrogen Corporation, USA. Goat anti-rabbit IgG conjugate, NC filter, protein molecular marker, and Taq DNA polymerase were obtained from Sino Promega Company, China. While the plasmid DNA extraction kit was obtained from Omega Bio Tek Corporation, USA. Bovine serum albumin, sodium deoxycholate, and calcium chloride were obtained from the Sangon Company, China. It was made sure that all of the biochemical reagents, obtained from different sources, were of the highest commercially available level of purification.
The New Zealand rabbits were given a mixture of 300mg purified AmPLA2 along with 1ml of PBS. These rabbits were subjected to bleeding for over 3 weeks after their primary immunization was done through 200mg of the enzyme, which was emulsified in an incomplete PBS (1ml). After that, an injection having 200mg of enzyme dissolved in 1ml of saline was administered for a continuous period of 3 weeks. A blood sample obtained was analyzed to detect the effect value of the serum given. It was done through native AmPLA2 after 1 week of the second dose of enhanced immunization, managed through gel diffused protocol as put forward by the Sambrook and Russell. Serum obtained was stored at a very low temperature of -80 degrees centigrade.
Intracellular proteins of infected cells, native AmPLA2, and normal insect cells were examined through 10% (v/v) SDS-PAGE, again according to the method described by Sambrook and Russell. The gels were stained with the Coomassie brilliant blue R250. Expressed fusion protein's concentration was evaluated through thin layer scanning of the SDS-PAGE gel. Unstained protein, separated, was transferred to unmodified NC membrane for Western blots. This process was carried out in a special Bio-Rad transblot set up. The fusion protein was examined with the help of a rabbit antiserum against the native AmPLA2. Proteins were detected on the blot through goat anti-rabbit IgG conjugate as prescribed by Sambrook and Russell.
PLA2's titrimetric assay was carried out by a revised method, previously used by Owen et al. Fresh egg yolk substrate, which was blended in 200ml of deionized and distilled water, was made fresh on daily basis. The egg mixture was subjected to a heat of 37 degrees centigrade and later NaOH was mixed in to maintain its pH value at 8.0. This process was done in a special thermostatted micro reaction vessel. PH stat titration was carried out through the pH meter system, which helped in keeping pH value at 8.0 after adding 0.005mol/L of NaOH. Solubilized protein extracts were measured carefully for their concentration of infected cells and later they were added to substrate mixture. Purified AmPLA2 and BV powder, which was diluted in deionized and distilled water were used a positive control while extracts of Tn cells were chosen for the negative control.
Result
EcoRI/Hind digested fragment of AccPLA2 (cDNA) from pGem- AccPLA2 was administered into transfer vector pFastBacHTa. Products of the registered digestion and the PCR of recombinant plasmid had a 400bp band – quite identical to AccPLA2 fragment. The nucleotide sequencing also confirmed that inserted fragment gives a correct ORF for protein expression. According to the schematic presentation of the recombinant transfer vector, the inserted gene was found to be under control of autographa californica nuclear polyhedrosis virus polyhedrin promoter. Expressed gene was naturally designed to establish a fusion protein that involves hexahistidine resides at the amino terminus that eventually allows single-step purification with immobilized Ni2+ ion affinity chromatography.
Once recombinant transfer vector pFastBacHTa – AccPLA2 was converted into E.coli DH10B capable cells, which contain the baculovirus shuttle vector bacmid, it was further cultured. PCR identification was then conducted with bacmid DNA that was isolated from positive colonies in the form of templates making use of primer PUC/M13 and AccPLA2, respectively. 2 fragments, one about 2700bp and the other 100bp were found. After examination, they were found to be like the AccPLA2 fragment within MCS (multiple cloning sites). On the other hand, a bacmid fragment of 2300bp was located within the left and right range of the bacterial transposon TN7 + AccPLA2 fragment. It confirms that rBacmid-AccPLA2 and recombinant bacmid had been accurately developed.
With the help of Lipofection, rBacmid-AccPLA2 DNA was made available in the Tn cells in medium. AccPLA2 showed in 3d after the infection of rBacmid-AccPLA2 DNA. Once the centrifugation was completed for generating cells, amplified rBacmid in the Tn cells, rTnV-Bac-AccPLA2, was used in supernatants for propagation. Once propagation was completed for the 4 generations, rTnV-Bac-AccPLA2 genomic DNA was used for performing PCR identification from infected cells. Agarose electrophoretic analysis depicted that PCR products had 400bp, which was identical to the AccPLA2 gene; it was hence confirmed that rTnV-Bac-AccPLA2 genomic DNA had been successfully propagated in the Tn cells.
Discussion
Before this clinical study, BVPLA2 had not been expressed in any insect cells. But here the AccPLA2 gene was expressed successfully in the Tn cells. It was established that the expressed fusion protein was found to be active, indicating that the Tn cells can express a properly modified AccPLA2. The findings of this clinical study are totally in harmony with the results of previous clinical reports - Soldatova et al, Altmann et al and Li et al. Glycosylation of the recombinant protein can be termed as an important mechanism that directly affects protein function. This study, in combination with the previous studies, can provide a scientific base for the molecular biological application of AccPLA2 in the future.
Conclusion
Tn cells were constructed by transfecting recombinant bacmid DNA, which was made a part of a new BVPLA2 gene through apis cerana cerana glands to help express recombinant AccPLA2 protein. It is a BVPLA2 type structure that has nearly the same level of biological activity; that is it can be easily glycosylated in the Tn cells. This clinical study can provide basic, but helpful information essential for engineering genetics that can be used for producing AccPLA2 in the pharmaceutical industry.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865836/