Efficient expression of porcine reproductive and respiratory syndrome virus genes using baculovirus system Zhou Yanjun, Qiu Huaji, Wang Yunfeng, Qian Ping, Cai Xuehui, Chai Wenjun, Weng Changjiang, Tong Guangzhi (State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) , Harbin 150001) DNA) co-transfected insect cells sf9, after three rounds of plaque purification, obtained recombinant baculovirus rBac_GP5. After infecting sf9 cells with this recombinant virus, the application of indirect immunofluorescence test, SDS_PAGE and Western blot detection showed that The GP5 gene has been highly expressed in the baculovirus system. Due to the difference in the degree of glycosylation, the expressed products have three apparent molecular weights of 22, 25 and 30 kDa, which account for about 26.4 of the total cellular protein.
Infectious diseases, such as reproductive disorders of pregnant sows (abortion, premature delivery, stillbirths and mummies), increased mortality of piglets, and respiratory diseases of pigs of all ages, are currently one of the important infectious diseases that threaten the healthy development of the pig industry. The pathogen is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which is a single-stranded positive-strand RNA virus with an envelope. Its genome size is 15 kb and contains 8 open reading frames (ORF PRRSV and equine arteritis virus (EAV), Monkey hemorrhagic fever virus (SHFV) and murine lactate dehydrogenase elevated virus (LDV) belong to the newly established Arteritis virus family, and the arteritis virus PRRSV has 6 structural proteins and 2 non-structural proteins. Among the corresponding authors, the glycosylated envelope protein (GP5 or E protein) encoded by ORF5 is one of the main structural proteins of the virus, but the structure and function of this protein are not very clear. Recent studies have shown that this protein It has 6 antigenic determinants, one of which is a serotype-specific linear neutralizing determinant, which can neutralize the infectivity of the virus in vitro. The virus neutralization has a significant correlation with the potency of the GP5 antibody. Plana and other rods The GP5 gene is expressed by the rhabdovirus, and the molecular weight of the expressed product is 17, 20, 23, and 25 according to the degree of glycosylation. Since the expressed product is in the form of a non-fusion protein, it is not conducive to isolation and purification. For further research Protein structure and function, and in order to facilitate purification and crystallization of the expression product, a fusion protein described herein expressed GP5 gene first isolated strain of PRRSV CH_la using a baculovirus expression system.
1 Materials and methods 1.1 The strain and the plasmid PRRSV CH_la strain were isolated from a pig farm in Beijing. It has been proven that the recombinant plasmid of the GP5 gene of the North American type PRRSV CH_la strain was constructed by this research team. Invit 1.2 Main Reagents A variety of restriction enzymes and modified enzymes were purchased from the transfection kit as Invitrogen products.
1.3 Construction of recombinant transfer vector plasmid The plasmid pUC18_ORF5 was digested with the restriction enzyme EcoRI, filled in, and then digested with PstI to recover the 638bp target fragment. At the same time, the pBlue BacHis B plasmid was digested with BamHI and filled in, digested with PstI, and then the target fragment of about 10 kb was recovered. Connect and transform in the usual way. The obtained recombinant plasmid was digested with PstI and identified by PCR (using the ORF5 specific primer P5S / P5R and the universal primer Bac1 / Bac2 provided by the transfection kit) and sequence determination. The recombinant transfer vector plasmid.
1.4 Screening of transfection and recombinant virus The transfection procedure was carried out according to the instructions of Invitro gen's transfection kit. X_gal was used as an indicator. After three rounds of plaque purification, recombinant baculovirus was obtained. The PCR identification of 1.5 recombinant viruses was performed using the universal primers Bac1 / Bac2 for PCR amplification. The circulation parameters were carried out according to the instructions of the transfection kit .
1.6 Expression and detection of recombinant baculovirus GP5 gene Infect sf9 cells with purified recombinant virus, harvest cells 96 hours after infection, prepare cell smears, fix with cold acetone, and perform indirect immunofluorescence detection with PRRS polyclonal serum . At the same time, sf9 cells from 24 to 96 hours after infection were taken for SDS_PAGE analysis, and PRRS polyclonal serum was used for Western blot analysis.
2 Results 2.1 Construction of recombinant baculovirus transfer vector The target fragment and the vector were digested, ligated and transformed, and the resulting recombinant transfer vector was identified by PstI digestion, and the specific primer P5S / designed according to North American VR_2332 strain P5R and baculovirus transfer vector universal primers Bac1 / Bac2 were identified by PCR, and the results were consistent with the expected results (Figures 1-3).
2. Recombinant transfer vector plasmid recombinant 4. Blank control (screening and purification of H2. 2 recombinant baculovirus) pBlueBacHisB_ORF5 and linear baculovirus DNA were co-transfected into sf9 cells, and after 3 days, the transfected cells were observed daily. Recombinant virus appeared as cytopathy but no polyhedron was formed. After three rounds of plaque purification, positive recombinant baculovirus was obtained.
2.3 PCR identification of recombinant baculovirus using recombinant baculovirus DNA as a template, PCR amplification using baculovirus universal primers Bac1 / Bac2, and setting up a transfer vector pBlueBacHisB_ORF5 as a positive control, the result was about 1. The 3kb fragment is the same size as the positive control. This indicates that the ORF5 gene of PRRSV has been recombined in the baculovirus genome (Figure 4).
Indirect immunofluorescence tests were performed on sf9 cells infected with recombinant virus for 96 hours. The results showed that compared with the cell control, the infected cells showed bright green fluorescence around, while the cell control showed a clear dark red. The sf9 fine GP5 gene recombinant virus was expressed in sf9 cells by infecting the recombinant virus at different times, and the molecular weight of the expressed fusion protein was 22kDa, 25kDa, and 30kDa. The content of the expression product was measured by thin layer scanning. Cell protein 1. Infection of sf9 cells for 96 hours 2. Infection of sf9 cells for 72 hours 3. Control of sf9 cells 4. Discussion of protein molecular weight standards Both the E protein expressed by baculovirus and the native E protein can be recognized by high PRRSV serum. Since the obtained recombinant fusion protein has 35 histidine residues (the molecular weight is about 5kDa), the molecular weights of the non-fusion E proteins expressed by the baculovirus are exactly the same and their glycosylation degrees are different, so the molecular weight is also Inconsistent, the sample belongs to the non-glycosylated form, while 30kDa and 25kDa belong to the completely glycosylated form. In addition, the total content of the fusion protein expressed by our baculovirus expression system accounts for about 26.4 of the protein content in the cell, which is much higher than the results reported by Plana et al. It may be related to the transfer vector or culture conditions.
Currently in eukaryotic expression systems, the baculovirus expression system has been widely used for its efficient and faithful expression. In recent years, with the further transformation of the baculovirus transfer vector, its transfection efficiency has been greatly improved. The transfer vector used in this research introduced the LacZ reporter gene, which made the screening of recombinant viruses easier and greatly improved the work efficiency. More importantly, the expression product is a fusion protein containing a polyhistidine tag, which can be separated and purified using a commercial affinity chromatography column, which greatly facilitates the post-processing of the expressed protein and is beneficial to a large number of Preparation or production.
With the intensive development of China's pig industry, PRRS has gradually become a major hidden danger. Although commercial inactivated vaccines and attenuated vaccines are available now, their immune efficacy and safety are unsatisfactory, and some vaccines have caused great controversy in their use. Therefore, it is necessary to develop new models with high immune efficacy and good safety. vaccine. In this study, the baculovirus system was used to express the PRRSV GP5 gene, which opened a way for the development of PRRS subunit vaccines, and also laid the foundation for further research on the function of viral structural proteins.
Chinese Journal of Preventive Veterinary Medicine A clone of a chicken Escherichia coli type and type pili protein structural gene Dai Dingzhen 1, Tang Houkuan 1, Zheng Mingqiu 2, Cai Baoxiang 2, Chen Puyan (1. Department of Animal Science, Hubei Agricultural College, Jingzhou 4332. Nanjing Agricultural University College of Veterinary Medicine, Nanjing 095) Two pairs of primers in the conserved region of papA. PCR amplified from a chicken pathogenic Escherichia coli (HJ20e) chromosome with a mannose-sensitive hemagglutination property (MSHA) and a mannose-resistant hemagglutination property (MRHA) to two species with sizes of 657bp and 541bp, respectively Positive product. Nucleic acid sequence determination confirmed that the amplified DNA fragments were pilA and papA genes respectively. After enzyme digestion, ligation, transformation and screening, positive clones of pilA and papA genes were obtained.
Chicken Escherichia coli has two types of fimbriae, namely type 1 and type P fimbriae, both of which are encoded by chromosomes. Type 1 fimbriae can play an adhesive role on chicken respiratory epithelial cells, which is conducive to bacteria colonization and reproduction in the host body [2, 3]. It is encoded by piA and related gene clusters. Most chicken pathogenic E. coli can express type 1 fimbriae. Type 1 fimbriae can mediate the specific adhesion of bacterial cells to many animal red blood cells, yeast cells and many other types of cells, but this adhesion can be inhibited by D_mannose. Because of this characteristic, type 1 fimbriae are also known as mannose-sensitive hemagglutinating properties (MSHA) fimbriae. P-type fimbriae are encoded by pap and related gene clusters, which are closely related to human, dog and pig urethral E. coli infections [4, 5]. P fimbriae can also agglutinate red blood cells, but only people with O-type blood Only red blood cells can make P-type fimbriae show good hemagglutination properties. Because its hemagglutination cannot be inhibited by mannose, it is also called mannose anti-hemagglutinating property (MRHA) fimbriae. P fimbriae are considered to be unable to directly adhere to the chicken tracheal mucosa epithelium, but it plays an important role in the further development of chicken colibacillosis. Type 1 fimbriae and P fimbriae have been regarded as important virulence factors for E. coli [1, 3]. Some bacteria can have both type 1 fibrillar and P fimbriae. Because fimbriae are a protein, some people have The pilin subunit vaccine is made, and after immunizing the chicken, it is found that it has very good protective effect. The single pilus is composed of more than 1,000 pilin protein subunits encoded by the pilA or papA genes. There have been no reports at home and abroad of the simultaneous cloning of structural genes of type 1 fimbriae and P fimbriae from the same chicken E. coli strain. There are only reports of simultaneous cloning of structural genes of type 1 and type P fimbriae from human E. coli. The study found that type 1 and type P fimbriae proteins between human and chicken E. coli have homology [2, 8]. This study intends to use human E. coli pili structural gene DNA sequence as a template, in its same Design primers in the source region, use PCR technology to simultaneously amplify and clone the structural genes of type 1 and type P fimbriae from chicken E. coli chromosome samples, and determine the nucleic acid sequence [6] Guo Baoqing, Chen Zhangshui, Liu Wenxing, etc. Chinese livestock and poultry infectious diseases, 1996, 2: 1-4.
[7] Qiu Huaji, Guo Baoqing, Tong Guangzhi, etc. Chinese Journal of Veterinary Medicine, 1998, 18 (2): ~ 121.
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