食品中弧菌免疫学筛检方法建立研究
摘要
食物中毒菌的检测一直是食品安全领域的热点问题,随着海水养殖业的蓬勃发展,由致病性弧菌造成的食物中毒病例频繁发生。现行的弧菌检测方法要么针对样品单一、耗时费力;要么检测试剂昂贵,不利于推广。因此,建立一种广谱快速、低耗高效、灵敏便捷的弧菌筛检方法尤为重要。本研究旨在找到弧菌属细菌的共同抗原,以此抗原为靶标,建立可同步筛检多种弧菌的免疫学检测方法。弧菌外膜蛋白位于弧菌最外层,具有与外界广泛接触的机会;弧菌鞭毛主要由鞭毛丝蛋白组成,抗原性好,保守性高。这些特点使它们成为弧菌属细菌共同抗原的首选目标。OmpK为弧菌噬菌体KVP-40的受体,可能存在于多种海洋弧菌的外膜之中;鞭毛基因FlaA、FlaB、FlaF编码弧菌极鞭毛丝蛋白基因,鞭毛丝蛋白保守性高。首先,本实验从弧菌OmpK的基因入手,通过克隆获得5株弧菌标准株OmpK基因并测序比较,从分子水平证明OmpK基因为多种弧菌所共有。同时,本实验成功构建副溶血弧菌多联鞭毛丝蛋白结构基因F,通过PCR克隆获得副溶血弧菌鞭毛基因片段(FlaA﹑FlaB﹑FlaF),用柔性Linker序列(Gly4Ser)进行串联,通过套叠PCR扩增出副溶血弧菌多联融合鞭毛基因F。其次,实验设计利用溶藻弧菌OmpK基因和多联融合基因F构建原核表达重组质粒pET22b-OmpK和pET22b-F,转化大肠杆菌BL21(DE3),在IPTG诱导下获得高效表达。之后,通过纯化两种重组蛋白分别免疫豚鼠和獭兔制备多抗血清,经ELISA检测两种多抗血清效价分别为1:51200和1:6400。两种多抗血清均可与多种弧菌发生反应而与非弧菌类食物中毒菌不识别,说明两种血清均具有较好的弧菌属特异性。利用纯化的多抗建立可同时筛选多种弧菌的抗原捕获ELISA方法;同时,利用重组表达的OmpK免疫BALB/c小鼠,制备抗外膜蛋白K的单克隆抗体,旨在筛选出具有弧菌属特异性的单克隆抗体,进而有针对性的定位弧菌属特异性的抗原表位,为以后的实验工作奠定基础。
Food safety is a capital problem at present. It has being paid more attention because the food is the material foundation for human’s life. Pathogenic Vibrio is the most common pathogen in food poisoning on littoral. Vibrio species are predominantly found in aquatic environments. While many of the species are thought to be harmless to humans.The genus Vibrio is represented by forty-six species. Twelve of them have been associated with human infections.Vibrio food poisoning which has high attack rate and huge perniciousness is diverging or fulminate on littoral. The most momentous problem is how to control the food poisoning from Vibrio. The existing detection methods are not accord with the society development. The species-specific gene of vibrio has been used to establish detection method in vibrio medical diagnosis or food sanitation probation. But sync-tacho-screening for varied vibrios according to genus-specific gene as a target has not been reported. Tacho-screening is more valuable than rigorous diagnose on acute food poisoning . In order to develop a sync-tacho-screening for varied vibrio diagnose, we discussed the feasibility of the utilization of ompK protein and poly-recombinant flagella gene F on vibrio diagnose which will become a novel method for the detection of bacterial in food
OmpK gene from five Vibrio species was cloned and analyzed
OmpK protein has chance to get in touch with outside because it is situated on the outer layer of bacterial. For research the homology of OmpK gene from Vibrio, we designed degenerate primers and obtained 800bp gene fragment of OmpK by the PCR amplification. The results showed that OmpK gene (GenBank accession nos. FJ462705-FJ462709) cloned from five Vibrio species contain ORF of 798-822 bases encoding 266-274 amino acids. And we compared the OmpK gene and predicted amino acid sequence with Vibrio OmpK gene logged in NCBI. Phylogenetic analysis of the OmpK proteins indicated that OmpK proteins belonged to the same protein family. Besides the first 20 amino acid residues of the singal peptide. OmpK proteins have some high similar domains from seventeen Vibrio species. It implied that the common epitope of the Vibrios maybe exist in these regions. Homologies of the OmpK proteins between V. alginolyticus and other Vibrio species were analyzed, which have a maximum identity of 99.2% compared to the OmpK protein of V. alginolyticus.
The OmpK gene from V. alginolyticus was chosen to over-expressed. It was linked to prokaryotic expression vector pET-22b. Effective expression from E. coli BL21(DE3) was induced by IPTG, which was contained recombinant plasmid pET-22b-OmpK. The optimal inducing concentration of IPTG was 1.0 mM and the optimal inducing time is 6 hours. SDS-PAGE showed that The molecular weight of recombinant OmpK proteins was about 28 kDa, which was less than 30 kDa of the molecular weight calculated previously.
Cloning and expression of poly-recombinant flagellar gene F from Vibrio parahaemolyticus
In this study, flagellae gene FlaA, FlaB and FlaF were cloned from the genome DNA of V. parahaemolyticus,by PCR amplification. Then they were linked to pMD18-T vector and were identified by restriction enzyme digestion. Then the obtained gene fragments were linked to gain cascaded peptide chain FlaA-FlaF-FlaB with determinate Linker sequence by overlap extension PCR. Restricted enzyme digestion sites Nde I and Xho I were added to the 5/ and 3/ of FlaA-FlaF-FlaB (F gene). The recombinant plasmids pMD18T-F were digested with NdeⅠand XhoⅠ, then they were linked to pET-22b vector digested with the same restriction enzymes. The recombinant plasmids pET22b-F confirmed by sequencing were transformed into E. coli BL21(DE3) for ultimate expression. The sequence results indicated that the gene mutation happened in 203, 355 and 837 position. The proper order were AGT→ATT, TAA→TGA and CTT→CCT, which had reading frame correctly and completely. Poly-recombinant flagellar gene F had 3420bp and coding 1140 amino acids. The molecular weight of poly-recombinant protein was 119 kDa predicted by DNAsis.
E. coli BL21(DE3) cells transformed with pET-22b-F plasmids were cultured in LB-ampicillin (100 mg/mL) of 1 L at 37℃. Protein expression was induced with a final concentration of 1 mM IPTG. The cells were cultured at 37℃for a further 4 h. The result of SDS-PAGE showed that the protein occupied 23.5% of the whole tropina. The expressed protein format was cytoryctes principally, and small quantity was soluble protein. Antigenicity analysis of OmpK and poly-recombinant flagellar protein
The purified recombinant proteins were quantified by UV method, and the concentration was adjusted to 1 mg/mL. Recombinant proteins were suspended (1:1) in Freund's complete adjuvant and were injected intraperitoneally into guinea pigs and rabbits. Before immunization, the animals were bled and their blood serums were collected as a negative control. Antibody titer of serum after the third immunity was detected by indirect ELISA. Antibody titer of the OmpK antiserum indicated that the best dilute multiple of correspond antibody was 1:51200 while the antigen dilution was 0.5 mg/L. Antibody titer of the flagellar protein antiserum was 1:6400 while the antigen dilution was 2 mg/L. The results showed that the recombinat protein had a evidently immunogenicity which expressed from prokaryotic expression system. Sensitivity and genus-specificity detection by ELISA
11 Vibrio strains had been used to detect the sensitivity of the antiserum by indirect ELISA. The optimal dilution of antiserum from rabbits was 400×. The sensitivity can reach the 103 cfu/mL. There were no positive reaction between the antiserum and other 27 kinds of non-vibrio intestinal infection bacteria tested by ELISA. The result indicated that the anti-flagellar protein antiserum has vibrio genus- specificity. The best dilution of anti-OmpK antiserum was 1:1600. The sensitivity can reach 103 cfu/mL. There were no positive reaction between the antiserum and other 27 strains non-vibrio strains tested by ELISA. The result indicated that the antiserum had vibrio genus-specificity which was better than anti-coalesce flagellar protein antiserum. Establishment of AC-ELISA for vibrios detetion
AC-ELISA was developed with the vibrio genus-specific polyclonal antibodies. ELISA plate was coated with antibody against OmpK at 4℃for 12h, and the optimal coating concentration was 2 mg/L. The primary polyclonal antibody was pruified from rabbits’serum, which against coalesce flagellar protein. The optimal working concentration was 1 mg/L at 37℃for 1h. After washing three times with PBST, 1% BSA was used as blocking at 37℃for 1h. The antibody with enzyme labelled was goat anti rabbit antibody with 1: 10,000 dilution at 37℃for 1h. OPD was used as coloration for 20 min.
Preparation McAb against OmpK protein of Vibrio alginolyticus
In order to locate the target site of Vibrio genus-specific antigen, the McAb against recombinant OmpK protein was prepared. Five McAbs cells were obtained by twice cell-fusions, which were named 2A7, 1A5, 1D7, 3G6 and 3H2. They can only reaction with V. alginolyticus OmpK protein except 2A7, which can react with V. alginolyticus and V. Parahemolyticus.
In a word, this study, firstly, demonstrated from molecular level that the OmpK protein were existed in varied Vibrios. Then, the recombinant OmpK protein of V. alginolyticus was over-expressed in E. coli BL21(DE3). The polyclonal antiserum against OmpK was also prepared. Then, the Vibrio OmpK protein has genus specificity from immunological level with polyclonal antiserum. Meanwhile, from another view, the poly-cecombinant flagellar protein of V. Parahemolyticus was expressed, which has high conservatism. The rabbit antiserum was prepared by immunization of flagellar protein F. Many kinds of food poisoning Vibrios can be recognized by two antiserums, which wouldn’t react with other non-vibrio. Moreover, the detected effect of anti-OmpK antiserum was better than anti-flagellar antiserum by comparison. Therefore, the AC-ELISA method foundation which utilized two kinds of polyclonal antibody to screen varied pathogenicity vibrios was useful to vibrio screening methods. We anticipate that this work will be helpful for facilitating future study of detection and protective vaccines of Vibrios.
Food safety is a capital problem at present. It has being paid more attention because the food is the material foundation for human’s life. Pathogenic Vibrio is the most common pathogen in food poisoning on littoral. Vibrio species are predominantly found in aquatic environments. While many of the species are thought to be harmless to humans.The genus Vibrio is represented by forty-six species. Twelve of them have been associated with human infections.Vibrio food poisoning which has high attack rate and huge perniciousness is diverging or fulminate on littoral. The most momentous problem is how to control the food poisoning from Vibrio. The existing detection methods are not accord with the society development. The species-specific gene of vibrio has been used to establish detection method in vibrio medical diagnosis or food sanitation probation. But sync-tacho-screening for varied vibrios according to genus-specific gene as a target has not been reported. Tacho-screening is more valuable than rigorous diagnose on acute food poisoning . In order to develop a sync-tacho-screening for varied vibrio diagnose, we discussed the feasibility of the utilization of ompK protein and poly-recombinant flagella gene F on vibrio diagnose which will become a novel method for the detection of bacterial in food
OmpK gene from five Vibrio species was cloned and analyzed
OmpK protein has chance to get in touch with outside because it is situated on the outer layer of bacterial. For research the homology of OmpK gene from Vibrio, we designed degenerate primers and obtained 800bp gene fragment of OmpK by the PCR amplification. The results showed that OmpK gene (GenBank accession nos. FJ462705-FJ462709) cloned from five Vibrio species contain ORF of 798-822 bases encoding 266-274 amino acids. And we compared the OmpK gene and predicted amino acid sequence with Vibrio OmpK gene logged in NCBI. Phylogenetic analysis of the OmpK proteins indicated that OmpK proteins belonged to the same protein family. Besides the first 20 amino acid residues of the singal peptide. OmpK proteins have some high similar domains from seventeen Vibrio species. It implied that the common epitope of the Vibrios maybe exist in these regions. Homologies of the OmpK proteins between V. alginolyticus and other Vibrio species were analyzed, which have a maximum identity of 99.2% compared to the OmpK protein of V. alginolyticus.
The OmpK gene from V. alginolyticus was chosen to over-expressed. It was linked to prokaryotic expression vector pET-22b. Effective expression from E. coli BL21(DE3) was induced by IPTG, which was contained recombinant plasmid pET-22b-OmpK. The optimal inducing concentration of IPTG was 1.0 mM and the optimal inducing time is 6 hours. SDS-PAGE showed that The molecular weight of recombinant OmpK proteins was about 28 kDa, which was less than 30 kDa of the molecular weight calculated previously.
Cloning and expression of poly-recombinant flagellar gene F from Vibrio parahaemolyticus
In this study, flagellae gene FlaA, FlaB and FlaF were cloned from the genome DNA of V. parahaemolyticus,by PCR amplification. Then they were linked to pMD18-T vector and were identified by restriction enzyme digestion. Then the obtained gene fragments were linked to gain cascaded peptide chain FlaA-FlaF-FlaB with determinate Linker sequence by overlap extension PCR. Restricted enzyme digestion sites Nde I and Xho I were added to the 5/ and 3/ of FlaA-FlaF-FlaB (F gene). The recombinant plasmids pMD18T-F were digested with NdeⅠand XhoⅠ, then they were linked to pET-22b vector digested with the same restriction enzymes. The recombinant plasmids pET22b-F confirmed by sequencing were transformed into E. coli BL21(DE3) for ultimate expression. The sequence results indicated that the gene mutation happened in 203, 355 and 837 position. The proper order were AGT→ATT, TAA→TGA and CTT→CCT, which had reading frame correctly and completely. Poly-recombinant flagellar gene F had 3420bp and coding 1140 amino acids. The molecular weight of poly-recombinant protein was 119 kDa predicted by DNAsis.
E. coli BL21(DE3) cells transformed with pET-22b-F plasmids were cultured in LB-ampicillin (100 mg/mL) of 1 L at 37℃. Protein expression was induced with a final concentration of 1 mM IPTG. The cells were cultured at 37℃for a further 4 h. The result of SDS-PAGE showed that the protein occupied 23.5% of the whole tropina. The expressed protein format was cytoryctes principally, and small quantity was soluble protein. Antigenicity analysis of OmpK and poly-recombinant flagellar protein
The purified recombinant proteins were quantified by UV method, and the concentration was adjusted to 1 mg/mL. Recombinant proteins were suspended (1:1) in Freund's complete adjuvant and were injected intraperitoneally into guinea pigs and rabbits. Before immunization, the animals were bled and their blood serums were collected as a negative control. Antibody titer of serum after the third immunity was detected by indirect ELISA. Antibody titer of the OmpK antiserum indicated that the best dilute multiple of correspond antibody was 1:51200 while the antigen dilution was 0.5 mg/L. Antibody titer of the flagellar protein antiserum was 1:6400 while the antigen dilution was 2 mg/L. The results showed that the recombinat protein had a evidently immunogenicity which expressed from prokaryotic expression system. Sensitivity and genus-specificity detection by ELISA
11 Vibrio strains had been used to detect the sensitivity of the antiserum by indirect ELISA. The optimal dilution of antiserum from rabbits was 400×. The sensitivity can reach the 103 cfu/mL. There were no positive reaction between the antiserum and other 27 kinds of non-vibrio intestinal infection bacteria tested by ELISA. The result indicated that the anti-flagellar protein antiserum has vibrio genus- specificity. The best dilution of anti-OmpK antiserum was 1:1600. The sensitivity can reach 103 cfu/mL. There were no positive reaction between the antiserum and other 27 strains non-vibrio strains tested by ELISA. The result indicated that the antiserum had vibrio genus-specificity which was better than anti-coalesce flagellar protein antiserum. Establishment of AC-ELISA for vibrios detetion
AC-ELISA was developed with the vibrio genus-specific polyclonal antibodies. ELISA plate was coated with antibody against OmpK at 4℃for 12h, and the optimal coating concentration was 2 mg/L. The primary polyclonal antibody was pruified from rabbits’serum, which against coalesce flagellar protein. The optimal working concentration was 1 mg/L at 37℃for 1h. After washing three times with PBST, 1% BSA was used as blocking at 37℃for 1h. The antibody with enzyme labelled was goat anti rabbit antibody with 1: 10,000 dilution at 37℃for 1h. OPD was used as coloration for 20 min.
Preparation McAb against OmpK protein of Vibrio alginolyticus
In order to locate the target site of Vibrio genus-specific antigen, the McAb against recombinant OmpK protein was prepared. Five McAbs cells were obtained by twice cell-fusions, which were named 2A7, 1A5, 1D7, 3G6 and 3H2. They can only reaction with V. alginolyticus OmpK protein except 2A7, which can react with V. alginolyticus and V. Parahemolyticus.
In a word, this study, firstly, demonstrated from molecular level that the OmpK protein were existed in varied Vibrios. Then, the recombinant OmpK protein of V. alginolyticus was over-expressed in E. coli BL21(DE3). The polyclonal antiserum against OmpK was also prepared. Then, the Vibrio OmpK protein has genus specificity from immunological level with polyclonal antiserum. Meanwhile, from another view, the poly-cecombinant flagellar protein of V. Parahemolyticus was expressed, which has high conservatism. The rabbit antiserum was prepared by immunization of flagellar protein F. Many kinds of food poisoning Vibrios can be recognized by two antiserums, which wouldn’t react with other non-vibrio. Moreover, the detected effect of anti-OmpK antiserum was better than anti-flagellar antiserum by comparison. Therefore, the AC-ELISA method foundation which utilized two kinds of polyclonal antibody to screen varied pathogenicity vibrios was useful to vibrio screening methods. We anticipate that this work will be helpful for facilitating future study of detection and protective vaccines of Vibrios.
引文
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