Skin Anaphylaxis Test of Purified Protein HAS1 in Cavies

Guoru XIONG Gengfeng ZHAO Jungang WANG Guilan XING Shuzhen ZHANG

Abstract The antifungal protein HAS1 is a new antifungal protein isolated from the genome of Bacillus subtilis HAS, which has a good inhibitory effect on various pathogenic fungi in sugarcane. This study aimed at evaluating the immunological transfer reaction of the test sample through repeated skin contact by observing whether the cavy skin repeatedly exposed to purified protein HAS1 suffers from allergic reaction and how strong is the allergic reaction. The results showed that the test group and the vehicle control group exhibited no allergic reaction in the skin immediately and at 24, 48 and 72 h and had an mean reaction score and a sensitization rate both of 0, so the results of the naked eye observation were both nonallergenic to cavy skin; and in the positive control group, the cavies were observed to be highly sensitized immediately after removing the drug, mildly sensitized at 24 h, and not sensitized at 48 and 72 h, which meant the skin allergy was alleviated with the observation time. It is suggested that the purified protein HAS1 is negative for cavy skin anaphylaxis test (nonallergenic to cavy skin), which provides an experimental basis for further utilization of the protein and its coding gene.

Key words Saccharum officinarum L.; Bacillus subtilis HAS; Purified protein HAS1; Cavies; Anaphylaxis

Sugarcane (Saccharum officinarum L.) is the most important sugar crop in China. Chinas sugarcane planting area is about 1.2 million hm2. Due to the lack of corresponding disease-resistant varieties, sugarcane diseases have become increasingly serious, which has become one of the main factors restricting the sustainable development of sugarcane industry and sugar enterprises. Breeding disease-resistant varieties is the most fundamental way to solve this problem, but conventional disease-resistant breeding takes a long time and is far from meeting the needs of production［1-7］. Therefore, using plant genetic engineering technology to introduce disease resistance genes into good sugarcane varieties for effective expression and realization of disease resistance is the most effective way to cultivate sugarcane resistant varieties.

The HAS1 antifungal protein is a new antifungal protein isolated from the genome of Bacillus subtilis HAS, which has a good inhibitory effect on various pathogenic fungi on sugarcane［8］. We intend to apply genetic engineering methods to introduce the HAS1 antifungal protein-coding gene into the main varieties for sugarcane production that are threatened by fungal diseases, thereby creating germplasm resources resistant to various fungal diseases.

The safety of genetically modified foods has always been the focus of attentionm, and we cant ignore this problem while carrying out plant genetic transformation. The acute toxicity test of purified protein HAS1 on KM mice has been already conducted［9］. In this study, the new antifungal protein HAS1 was tested on cavy skin for anaphylaxis according to relevant national regulations, providing an experimental basis for further utilization of the protein and its coding gene.

Materials and Methods

Test drugs and animals

The purified protein HAS1 for expression was obtained by expression and purification in laboratory. The vehicle control and the positive control were prepared in the laboratory of the Safety Evaluation Center or purchased. The cavy germ line was albino HD cavy (Hartley cavy), purchased from Changsha Tianqin Biotechnology Co., Ltd. The cavy feed was provided by Beijing Keao Xieli Feed Co., Ltd.

Test animal grouping and administration method

Thirty cavies that had a body weight meeting the standard and had undergone quarantine were selected and weighed in the range of 254.1-415.2 g. They were randomly divided into the test group, vehicle control group and blank control group, with 10 cavies in each group, half male and half female. The log sheet of the grouped caviesskin anaphylactic tests was filled. In this test, the concentration of the test solution was directly used as the sensitization and challenge concentration, and the sensitization and challenge doses were both 0.1 ml/cavy.

Index observation method

The operational procedures given by China Food and Drug Administration were followed［10-14］. The cavy sensitization was performed on days 0, 7 and 14 by applying the drug locally on the same area in the same way. After the drug was applied, it was covered with a layer of weighing paper and two layers of sterilized gauze, and fixed with non-irritating rubberized fabric. The drug in the smeared area was washed with warm water 6 h later, followed by filling the "Log sheet for active skin anaphylaxis test of cavies". The test was performed for a total of 3 times. For the positive control group, 1% (g/ml) 2,4-dinitrochlorobenzene was given, and the treatment method was the same as above.

The challenge contact was performed at 14 d after the last sensitization. The test sample, vehicle and positive drug 0.1% (g/ml) 2,4-dinitrochlorobenzene were applied to the right dehaired areas of animals in each group according to the method the same as sensitization. The drugs in the smeared areas were washed with warm water, and observation was performed immediately to see whether skin allergy reaction happened. The observation of skin allergic reaction was performed again at 24, 48 and 72 h, and the "Log sheet for challenge contact of cavies after active skin anaphylaxis test" was filled and photographed.

Data processing methods

All experiments were commissioned by the Hainan Provincial Drug Safety Evaluation Research Center. Animal body weight data were analyzed by ANOVA with SPSS 20.0 statistical software, and the differences in body weight between the test group and the vehicle control group were compared.

Results and Analysis

Observation of general symptoms

There were no abnormalities in general symptom observation and eating and drinking in each group during the experiments.

Visual observation of the administration area

The responses of the three groups during the sensitization exposure on days 0, 7 and 14 were observed. The test group and the vehicle control group showed no skin allergic reaction symptoms such as erythema and edema in the three times of sensitization exposure. For the positive control group, at 6 h after the first sensitization, 8 cavies showed mild erythema reaction and 2 cavies showed moderate erythema reaction, and these 10 cavies were accompanied by mild edema reaction at the same time; at 6 h after the second sensitization, 10 cavies showed mild erythema reaction, among which 6 cavies was accompanied by mild edema reaction, and the remaining 4 was only accompanied by edema reaction; and in the observation 6 h after the third sensitization, 10 cavies showed mild erythema reaction, and these 10 cavies were all accompanied by mild edema reaction. Fourteen days after the last sensitization, the right dehaired area was challenged, and after 6 h of application, the drug in the smeared area was washed with warm water. The skin allergic reaction was observed immediately and at 24, 48 and 72 h (as shown in Table 1).

No skin allergic reaction was observed in the test group and the vehicle control group immediately after the removal of drugs, and at 24, 48 and 72 h, and the mean score and sensitization rate were both 0, which meant that the cavy skin was free of allergy. As to the positive control group (given 2,4-dinitrochlorobenzene), seven (70%) cavies were immediately observed with mild and moderate erythema reaction, and seven with mild edema reaction, indicating a mean reaction score of 1.7 and a sensitization rate of 70%, so the drug was highly allergenic to cavies; at 24 h, two (20%) cavies were observed to have a mild erythema reaction, indicating a mean score of 0.2 and a sensitization rate of 20%, which meant that the cavies were mildly sensitized; and at 48 and 72 h, no symptoms of allergic reaction such as erythema and edema were observed, the mean reaction score and sensitization rate were both 0, so there was no allergenicity on cavies.

Discussion and Conclusions

There have been many successful reports on the operation of transgenic technology in sugarcane, including antiviral［15-18］, antibacterial［19］, pest resistance［20-21］, anti-nematode［22-23］, herbicide resistance［24-27］, drought resistance［27-28］and other aspects. The encoding gene of the purified protein in this study provided the antigenic material for this engineering operation, so it is necessary to carry out preliminary research.

In addition, the safety evaluation of genetically modified foods is one of the hot issues that people pay attention to. The genetic safety evaluation is usually referred to the safety evaluation of genetically modified crops. This study is characterized by the toxicologic tests on the obtained gene-encoded proteins prepared for transgenic technology operation, in order to provide an experimental basis for subsequent work.

In this study, the purified HAS1 protein was collected and applied to cavies for 3 times at days 0, 7 and 14, and challenge contact was performed at 14 d after the last sensitization. The applied areas were observed for skin allergic reaction immediately and at 24, 48 and 72 h after drug removal, and the results were compared with the vehicle control group. It was found that the skin allergy effect was equivalent, and the results of the naked eye observation were both allergenic to cavy skin. In the positive control group, the cavies were observed to be highly sensitized immediately after removing the drug, mildly sensitized at 24 h, and not sensitized at 48 and 72 h, which meant the skin allergy was alleviated with the observation time. The vehicle control was visually nonallergenic to cavy skin at the above time points. This result provides a reference for the further application of the purified protein HAS1 and its coding gene.

References

［1］LU GD, LI CC, PAN CZ, et al. China sugarcane disease list［J］. Sugarcane, 1997, 4(4): 19-23. (in Chinese)

［2］HUO XJ, LI CS, LU RS. Investigation and identification of sugarcane diseases in Guangxi［J］. Journal of Anhui Agricultural Sciences, 2013, 41(10): 4368-4369. (in Chinese)

［3］HUANG YK, LI WF. Colored atlas of main diseases, insect pests and weeds of modern sugarcane［M］. Beijing: China Agriculture Press, 2011. (in Chinese)

［4］AN YX, GUAN CX. Atlas of sugarcane pests and diseases and control［M］. Guangzhou: Jinan University Press, 2009. (in Chinese)

［5］LI ZP, ZHANG SZ. Atlas of sugarcane pest and disease diagnosis in Hainan［M］. Beijing: China Agriculture Press, 2014. (in Chinese)

［6］XIONG GR, LI ZP, ZHAO TT, et al. Primary investigation to sugarcane on the diseases in Hainan Province［J］. Chinese Journal of Tropical Crops, 2010, 31(9): 1-8. (in Chinese)

［7］PENG SG. Sugarcane breeding［M］. Beijing: Agriculture Press, 1990. (in Chinese)

［8］LIU HQ, HUANG YT, DAI MY, et al. Identification of in-intro antagonistic activity of HAS1 protein against Sporisorium scitanminea［J］. Agricultural Biotechnology, 2018, 7(06): 9-11,15.

［9］XIONG GR, ZHAO GF, WANG JG, et al. Study on acute toxicity of purified protein HAS1 expression in KM mice［J］. agricultural biotechnology, 2019, 8(4): 69-71.

［10］China Food and Drug Administration. Drug registration management measures, 2007. (in Chinese)

［11］China Food and Drug Administration. Quality management regulations for non-clinical research, 2017. (in Chinese)

［12］China Food and Drug Administration. Drug Research Supervision and Management Measures (Trial), 2005. (in Chinese)

［13］China Food and Drug Administration. Technical guidelines for single-dose toxicity studies, 2014. (in Chinese)

［14］China Food and Drug Administration. Guidelines for non-clinical safety evaluation of therapeutic biological products, 2007. (in Chinese)

［15］SMITH GR, GAMBLEY RL. Progress in development of a sugarcane meristem transformation system and production of SCMV-resistant transgenics［J］. Sugarcane, 1994, (6): 22.

［16］SMITH RG, JOYCE PA, HANDLEY JA, et al. Genetically engineering resistance to sugarcane mosaic and feiji disease viruses in sugarcane［C］//Sugar 2000 Symposium: Sugarcane: research towards efficient and sustainable production, 1996, 12: 138-140.

［17］IVAN L INGELBRECHT, JAMES E IRVINE, T ERIK MIRKOV. Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome［J］. Plant Physiology, 1999, 119: 1187-1198.

［18］YAO W, YU AL, XU JS, et al. Analysis and identification for transgenic sugarcane of ScMV-CP gene［J］. Molecular Plant Breeding, 2004, 2(1): 13-18.

［19］ZHANG LH, XU JL, ROBERT G BIRCH. Engineered detoxification confers resistance against a pathogenic bacterium［J］. Nature Biotechnology, 1999, 17(10):1021-1024.

［20］ARIEL ARENCIBIA, ROBERTO I VAZQUEZ, DMITRI PRIETO, et al. Transgenic sugarcane plants resistant to stem borer attack［J］. Molecular Breeding, 1997, 3(4): 247-255.

［21］ZHANGSUN DT, LUO SL, CHEN RK, et al. Improved agrobacterium-mediated genetic transformation of GNA transgenic sugarcane［J］. Section Cellular and Molecular Biology, 2007, 62(4): 386-393.

［22］JO-ANNE CH TAN, MICHAEL GK JONES, JOHN FOSU-NYARKO. Gene silencing in root lesion nematodes (Pratylenchus spp.) significantly reduces reproduction in a plant host［J］. Experimental Parasitology, 2013, 133(2):166-178.

［23］CHEN PH, XU LP, CHEN RK. Construction of expression vector of nematode-resistant gene and transformation of sugarcane［J］. Chinese Journal of Eco-Agriculture, 2004, 12(4): 57-59.

［24］GIL A ENRIQUEZ-OBREGON, ROBERTO I VAZQUEZ-PADRON, DMITRI L PRIETO-SAMSONOV, et al. Herbicide-resistant sugarcane (Saccharum officinarum L.) plants by agrobacterium-mediated transformation［J］. Planta, 1998, 206: 1367-1374.

［25］FALCO MC, TULMANN NETO A, ULIAN EC. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane［J］. Plant Cell Reports, 2000, 19(12): 1188-1194.

［26］MANICKAVASAGAM M, GANAPATHI A, ANBAZHAGAN VR, et al. Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds［J］. Plant Cell Rep, 2004,23(3):134-43.

［27］ZHANG SZ, ZHENG XQ. Gene gun-mediated genetic transformation of sugarcane［J］. Chinese Journal of Tropical Crops, 2001, 22(1): 35-41.

［28］ZHANG SZ, ZHENG XQ, LIN JF, et al. Cloning of trehalose synthase gene and transformation into sugarcane［J］. Chinese journal of agricultural biotechnology, 2000, 8(4): 385-388.