Comparative examination of susceptibility of two genotypes of carp (Cyprinus carpio L.) to infection with Aeromonas salmonicida

Progeny of a Hungarian breed as well as its F1 cross with the Japanese coloured carp were grown in ponds for 3 months. After artificial infection with Aeromonas salmonicida, susceptibility of the fish to carp erythrodermatitis was evaluated. Growth and condition factor of the hybrid carp were significantly higher than those of the homozygous group. Following the infection with Aeromonas salmonicida, morbidity of the hybrid was half that of the homozygous fish. The relative mass of spleen, kidney and head kidney of the fish having no clinical sign of infection was significantly higher than in the specimens with inflammatory or ulcerous local reactions. Non-susceptibility is presumably correlated with relatively extensive leucocyte propagation in the lymphoid tissues.     

Furunculosis in Atlantic salmon (Salmo salar L.) is not readily controllable by bacteriophage therapy

The potential of bacteriophage therapy to control bacterial disease in farmed fish was tested using, as an example, furunculosis of Atlantic salmon, caused by Aeromonas salmonicida subsp. salmonicida.

In vivo testing with Atlantic salmon and rainbow trout (Oncorhynchus mykiss Walbaum) showed no adverse effects, with bacteriophage generally cleared within 96 h of administration by either intraperitoneal (i.p.) injection or oral in-feed.

Juvenile Atlantic salmon were administered a combination of bacteriophage O, R and B (1.9 × 10⁸ pfu fish^− 1) by i.p. injection, after they had been challenged with A. salmonicida subsp. salmonicida 78027, also by i.p. injection. The fish that were injected with bacteriophage immediately after challenge died at a significantly slower rate then those that were either not treated with bacteriophage, or treated 24 h post-challenge. However, the end result (100% mortality) was not affected.

In further experiments the effects of oral (1.88 × 10⁵ pfu g^− 1 fish^− 1 daily for 30 days), bath (1.04 × 10⁵ ml^− 1 daily for 30 days) and i.p. (6.25 × 10⁷ pfu fish^− 1) phage treatment to control furunculosis in experimentally infected Atlantic salmon were compared with antibiotherapy (treatment with 10 mg kg^− 1 bw^− 1 day^− 1 oxolinic acid for 10 days), using an indirect cohabitation challenge. No protection was offered by any of the bacteriophage treatments, compared to the positive challenge group, although significant protection was offered by the oxolinic acid treatment. Analysis of samples taken from the trials demonstrated that bacteriophage were correctly administered to the fish and, on occasion, were isolated from fish that had succumbed to furunculosis. It was also shown that bacteriophage resistant A. salmonicida subsp. salmonicida isolates could be recovered from mortalities in all the treatment groups.

The results suggest that, although there were no safety problems associated with the approach, furunculosis in Atlantic salmon is not readily controllable by application of bacteriophage.     

Protection, immune response and side-effects in European whitefish (Coregonus lavaretus L.) vaccinated against vibriosis and furunculosis

As indicated by market demand and cultivation prospects, the whitefish (Coregonus lavaretus L.) has the potential to become an important alternative to rainbow trout in fish farming in Finland. The fish processing industry has called for a fish species having non-pigmented flesh, and there is a long tradition of whitefish farming for stocking purposes in Finland. However, cultivation in net cages in the brackish water of the coastal area exposes fish to vibriosis (Listonella (Vibrio) anguillarum) and furunculosis (Aeromonas salmonicida salmonicida). Hence, profitable whitefish farming requires the efficient control of both of these diseases. The efficacy of vaccination (Apoject 1800®) was studied in the laboratory using challenge tests and by monitoring specific antibody production (ELISA) and the blood leucocyte pattern. The fish (22 g) were vaccinated at 15°C and the immunity developed during 8 weeks prior to challenge with vibriosis (1.8×10⁵ cfu) and 12 weeks prior to challenge with furunculosis (4.9×10² cfu). The challenge was performed by i.p. injection. Side-effects around the injection site were studied 16 weeks post-vaccination. The immune system of the whitefish responded well and vaccination gave good protection against vibriosis and furunculosis. The RPS was 100% following the Vibrio challenge and 99% following the Aeromonas challenge. The antibody levels continued increasing in the vaccinated group throughout the study period. Lymphocyte and neutrophil counts were elevated in vaccinated compared to non-vaccinated fish at 8 weeks post-vaccination, but not after 16 weeks. Vaccination produced weak adhesions around the injection site in 42% and minor pigmentation in 9% of the 65 fish examined.     

Passive immunisation of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes

Passive immunisation of fish was conducted to determine whether anti-Vibrio anguillarum whole sera (AVA) and affinity-purified AVA raised in sheep, rabbits and rainbow trout (Oncorhynchus mykiss) were persistent when injected and orally administered into rainbow trout. These responses were compared with active immunisation by immersion in, and intraperitoneal (i.p.) injection with, formalin-killed V. anguillarum cells. Sheep and rabbit AVA were detected in rainbow trout sera for up to 70 days (half-life 21 days) after i.p. injection as determined by an enzyme-linked immunosorbent assay (ELISA). The relative percentage survival (RPS) of passively immunised rainbow trout challenged with virulent V. anguillarum after an injection was comparable to that of active immunisation by immersion after 1 month post-immunisation (p.i). Affinity-purified sheep and rabbit AVA exhibited the same protective potential as whole serum in rainbow trout. Rabbit and sheep immune sera diluted 1:8 and 1:50, respectively, provided equivalent protection as undiluted fish immune serum. An active immune response against passively acquired heterologous immunoglobulins was demonstrated by ELISA, with responses against sheep AVA being less than those against rabbit AVA. Rainbow trout given purified sheep AVA conjugated to LTB (the GM-1-binding subunit of Escherichia coli heat-labile toxin) and administered orally had an RPS of 37.5% at 15 days and 27% at 1 month p.i. In contrast, fish given sheep AVA conjugated to TraT (an internal membrane of E. coli) or in micellar form with Quil-A had RPSs of only 18.7 and 6.2%, respectively, after 15 days, and 13.3 and 0% after 1 month, respectively. The protection conferred by immune sera was shown to be due to the immunoglobulin component alone. Heat inactivation of the complement in sera had no effect on the potency of immune sera.     

Assay performance during validation of freezing channel catfish Ictalurus punctatus (Rafinesque) infected with a Gram‐negative bacterium

Recovery of bacteria from infected fish during population sampling can be affected by factors including the type of assay, method of specimen preservation and concentration of bacteria present. Consequently, before use in field sampling, methods should be validated. The three objectives of this study were, first, to determine whether a channel catfish Ictalurus punctatus (Rafinesque) fingerling classified as positive for Gram‐negative Edwardsiella ictaluri infection according to bacterial culture before freezing was also classified as positive after freezing, second, to determine how direct culture from the kidney (DIRECT), culture of homogenate (HOMOG) and standard PCR (PCR) agree with bacterial culture in terms of classifying fish as positive or negative and third, to estimate diagnostic sensitivity (dSe) and diagnostic specificity (dSp) for DIRECT, HOMOG and PCR. In fresh and frozen fish, as bacterial concentration decreased, the ability of each assay to detect positive fish also decreased, especially when there were <10⁴ colony‐forming units per gram (CFU g^?1) tissue. HOMOG proved to be the most reliable at correctly classifying catfish, whether they were subclinically or clinically infected. PCR assay was the least reliable. Overall, values for this study population for dSe were 0.66, 0.92 and 0.43, and for dSp were 0.86, 0.91 and 0.95, for DIRECT, HOMOG and PCR respectively.     

Bactericidal activity of cecropin B and cecropin P1 expressed in fish cells (CHSE-214): application in controlling fish bacterial pathogens

Cecropins are a group of antimicrobial peptides which have bactericidal activity against a broad range of bacteria. To date, the cecropins used in a variety of studies were either purified from their natural source or obtained by chemical synthesis. The present study was conducted to test whether bactericidally active cecropins could be expressed in a fish cell line. For this purpose, Chinook salmon embryo cells (CHSE-214) were transfected with cecropin transgene constructs: Hyalophora cecropia preprocecropin B, procecropin B, cecropin B, and porcine P₁ cecropin. From the transfected cells, single cell clones were selected and screened for the presence of cecropin gene constructs by PCR amplification. The expression of the cecropin transgene in the PCR positive clones was determined by RT-PCR reaction. Southern blot hybridization results showed that the cecropin gene constructs were integrated into the genome in a multiple integration pattern. Bactericidal activity of the cecropins, synthesized from transgene constructs, was detected using inhibition zone assay for fish pathogenic bacteria: Aeromonas hydrophila, Pseudomonas fluorescens, and Vibrio anguillarum. Cecropin antimicrobial peptides produced in CHSE-214 cells possess bactericidal activity against these three fish pathogenic bacteria.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated.     

Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection

A susceptibility study of postlarvae (PL) and juvenile Super Shrimp®, a selected line of Penaeus stylirostris, was conducted to compare their resistance to infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection to that of a specific pathogen free (SPF) population of P. vannamei. Super Shrimp® PLs were fed with IHHNV-infected shrimp tissue for 2 days and then maintained on a pelletized ration for an additional 28 days. PLs were sampled at days 0, 1, 2, 3, 4, 6, 10, 15, 20, 25 and 30. There was no apparent mortality during the experimental period. Tissue DNA extracted from the PLs was analyzed for the presence of IHHNV by PCR. Low levels of IHHNV were detected only in DNA extracts from samples at days 1, 2, and 3. No IHHNV DNA was detected from days 4 to 30. The days that the PLs were weakly IHHNV-PCR positive were during the period that they were being fed with IHHNV-tissue, and thus, the IHHNV DNA signal was suspected to be from the infected tissue used as a feed. Through both histology and in situ hybridization, we confirmed that tissues of Super Shrimp® PLs were not infected with IHHNV. PCR results of another IHHNV challenge study with juveniles of Super Shrimp® were similar to those with PLs. These results indicate that IHHNV did not replicate in the PL and juvenile Super Shrimp®. In contrast, P. vannamei juveniles, which were used as a positive control, showed a more intense IHHNV infection, as determined by PCR detection, beginning at 6 days postchallenge and increasing throughout the remainder of the study. In addition, the IHHNV-infected P. vannamei at 30 days postchallenge showed histological changes characteristic of IHHNV infection and had a positive reaction for IHHNV with in situ hybridization. Our studies show that Super Shrimp® are resistant to IHHNV infection. This is the first unequivocal demonstration of resistance to viral infection in shrimp.     

Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.     

Two microalgae Crypthecodinium cohnii and Phaeodactylum tricornutum as alternative source of essential fatty acids in starter feeds for seabream (Sparus aurata)

Despite oils extracted from algae and other microorganisms that may constitute excellent sources of HUFAs, few studies have determined the nutritional value of different microalgal species for young marine fish. Six thousand gilthead seabream (Sparus aurata) postlarvae (73 mg body weight) were fed for 57 days diets containing either fish oil as a single lipid source or 2 and 4% of Cryptecodinum cohnii or 5% Phaeodactylum tricornutum. Fish oil substitution by C. cohnii resulted in improved fish survival and a very good growth performance, in agreement with a higher proportion of DHA in diets and in total lipids of fish. Incorporation of DHA and other fatty acids was proportional to their contents in diet suggesting the good nutritional utilization of homogenized C. cohnii. Lower survival rates were found in fish fed P. tricornutum and could be related to an epithelial degeneration observed in the anterior intestine. This degeneration could be related to a higher lipid content in these fish or to the strong hornlike cornutate processes found in the valves of the diatom P. tricornutum.     

Infections of nervous necrosis virus in wild and cage‐reared marine fish from South China Sea with unexpected wide host ranges

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage‐reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage‐reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage‐reared fish, respectively. However, by nested RT‐PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage‐reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage‐reared fish by seminested PCR assay, respectively. However, by nested RT‐PCR, the positive signal was observed in 65.2% and 100% species of wild and cage‐reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red‐spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty‐five species of the marine fish were the new hosts of NNV.     

Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans

The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.

Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.

In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.

Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper.     

Comparison of two assay methods for determination of nutrient and energy digestibility in fish

Determination of digestible energy (DE) and digestibility of protein, lipid and nitrogen-free extract (NFE) was studied by two methods in rainbow trout held in fresh water. The whole diet-substitution method used a reference diet composed of casein, dextrinised maize starch and fish oil supplied with minerals and vitamins. Test samples of fish meal were substituted for the reference diet at 25.0, 37.5, 50.0 and 75.0% in the test diets. Energy values and nutrient digestibilities of test samples were calculated from the values determined for the reference and the test diets using the substitution principle. The ingredient-substitution method used the same reference diet but in the test diets, test samples of fish meal were substituted for casein at levels of 15.0, 25.0, 30.0, 45.0 and 50.0%. Energy values and protein digestibility of the casein were determined in initial studies and used together with those of the reference diet to determine nutrient and energy digestibilities of the test samples. Thus, in the whole diet-substitution method, the digestibility of the test substance was determined relative to that of the reference diet, while in the ingredient-substitution method the values are relative to those of casein.

The study revealed no major differences between the two methods with regard to determined values and variability, and for both methods nutrient and energy digestibilities could be determined with a high level of accuracy. The ingredient-substitution method revealed a positive effect of fish meal on NFE digestibility, leading to a slight overestimation of the DE in fish meal that decreased when levels of inclusion increased.

For the whole diet-substitution method, inclusion of fish meal reduced the level and increased the digestibility of NFE in the test diets compared with that of the reference diet due to dilution, and consequently the determined DE value of the diets increased, which in turn led to an overestimation of the DE value of the fish meal. Since the casein-based ingredient-substitution method is based on a reference substance of presumably constant composition and digestibility, this method allows for comparison of results over time within the same laboratory as well as between laboratories, and may therefore be recommended.     

Detection of tetracycline-resistance determinants by multiplex polymerase chain reaction in Edwardsiella tarda isolated from fish farms in Korea

To determine and discriminate the types of tetracycline (Tc)-resistance determinants (tet), we developed a multiplex polymerase chain reaction (PCR). With minimized numbers of primers derived from the variable and conserved regions of six different types of tet genes, tet(A)–(E) and (G), the multiplex PCR was sensitive and specific enough to discriminate the various types of tet genes, even multiple tet genes in an individual resistant isolate, by the different sizes of the resulting PCR products. Each of 20 Tc-resistant Edwardsiella tarda (Ed. tarda) isolates from diseased fish from aquatic farms in Korea carrying either one or two tet genes of types (A), (D), (B), or (G) gave PCR products of the appropriate lengths. Among the four types of tet genes found in Ed. tarda, two types, tet(A) and (D), were always present on mobile plasmids, and the other two types, tet(B) and (G), were located on the nonmobile nucleic acids. This is the first time that either of these two genes, type (B) or (G), have been found in Ed. tarda isolates. The two most common types of Tc-resistance determinant were, tet(A) and (D), occurring in 55% and 45% of total Tc-resistant Ed. tarda isolates, respectively.     

Preliminary estimation of tropical ornamental fish metabolite production rates

The rational design of closed aquaculture systems requires data on metabolite production rates. The metabolite of primary concern is often ammonia. Ammonia formation rates for a number of tropical ornamental fish types and sizes were determined. Cyprinus carpio, Barbus conchonius, Pseudotropheus auratus and Carassius auratus auratus were used as representative egg-layers while Xiphophorus variatus and Xiphophorus helleri represented the live-bearers. Specific metabolite production rates, R′, were determined not only in terms of mass of fish but also the amount of feed. For instance, R′ (Amm-N) for 1.33 g unit size Barbus conchonius was determined to be 155.9 mg Amm-N kg-fish⁻¹% feed⁻¹ day⁻¹. In an attempt to produce a general model for all the ornamental fish investigated, all values of R′ obtained were correlated with fish sizes to produce an equation R′ (Amm-N) = 1.57-[30×log(itUW)] where UW = unit fish size.     

Safety and efficacy of emamectin benzoate administered in-feed to Atlantic salmon, Salmo salar L., smolts in freshwater, as a preventative treatment against infestations of sea lice, Lepeophtheirus salmonis (Krøyer)

The safety and efficacy of emamectin benzoate, administered in-feed to Atlantic salmon smolts, Salmo salar L., held in freshwater, was evaluated as a preventative treatment against sea lice, Lepeophtheirus salmonis, following transfer of fish to seawater.

In the safety study, salmon smolts held in freshwater were fed with diets containing emamectin benzoate at nominal doses of 0 (control), 50 (recommended dose) and 250 (5× recommended dose) μg kg⁻¹ fish day⁻¹ for 7 days (days 0–6). Actual dose rates, based on measured concentrations of emamectin benzoate in feed, differences in fish weight, and feed consumed, were 0, 54, and 272 μg kg⁻¹ day⁻¹, respectively. On day 9, fish were transferred to seawater and observed for 14 days. No differences in feeding response, coordination, behaviour, gross and histological appearance were observed between control fish and those that received 54 μg kg⁻¹ day⁻¹. Among smolts that received 272 μg kg⁻¹ day⁻¹, approximately 50% exhibited darker coloration, and one fish (1%) exhibited uncoordinated swimming behaviour. No pathognomonic signs of emamectin benzoate toxicity were identified.

In the efficacy study, smolts held in freshwater were fed an unmedicated ration (control group) or emamectin benzoate at 50 μg kg⁻¹ day⁻¹ (treated group) for 7 days (days 0–6). On day 9, fish were re-distributed to eight seawater tanks, each holding 30 control and 30 treated fish. On days 28, 56, 77 and 109, respectively, control and treated fish in two tanks were challenged with L. salmonis copepodites. When lice in each group reached chalimus stage IV, fish were sampled and the numbers of lice were recorded. Fish challenged at day 109 were sampled for the second time when lice were at the adult stage. Efficacy was calculated as the reduction in the mean number of lice on treated fish relative to the mean on control fish. Treatment with emamectin benzoate resulted in an efficacy of 85.0–99.8% in fish challenged at days 28–77, from the start of treatment, and lice counts were significantly lower (P<0.001) on treated fish than on controls. When fish challenged at day 109 were sampled at day 128, efficacy was 44.3%, but survival of chalimus to adult lice on treated fish was lower, and at day 159, efficacy had increased to 73%. These results demonstrate that treatment of salmon smolts with emamectin benzoate in freshwater was well tolerated and highly effective in preventing sea lice infestation following transfer of fish to seawater.     

抗冻蛋白AFPⅢ的原核表达、纯化及多克隆抗体的制备

采用基因重组技术将AFPⅢ基因与原核表达载体pET32a(+)连接,转化大肠杆菌BL21(DE3),通过PCR、单双酶切及测序鉴定构建结果.用IPTG诱导蛋白表达,将融合蛋白纯化后,免疫6-7周龄的小白鼠,制备AFPⅢ多克隆抗体,采用Western印迹法检验抗体特异性.通过酶联免疫吸附试验(ELISA)测定多克隆抗体滴度.成功地构建了AFPⅢ的原核表达载体,经在大肠杆菌中诱导表达、镍柱亲和层析纯化,得到较纯的相对分子质量约26 000 Da的融合蛋白,免疫小白鼠后得到多抗血清,Western印迹结果显示此多克隆抗体与AFPⅢ蛋白特异性结合.该试验为进一步研究AFPⅢ在转基因鱼中的定点表达以及作用机制奠定了基础.