Algae feed capable of preventing avian influenza and application thereof
A technology of avian influenza and Chlamydomonas lychee, applied in the field of algae feed, can solve the problem of the absence of avian influenza antigen genes
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Embodiment 1
[0059] [Example 1] Optimization and amplification of avian influenza antigen genes
[0060] According to the known avian influenza antigen genes (GenBank accession number: AY609312) and the codon preference of Chlamydomonas reinhardtii, the codons that can be preferentially expressed in the nucleus of Chlamydomonas reinhardtii are selected to replace the known avian influenza antigen genes , and the amino acid sequence remains unchanged, thereby obtaining the HAV1 and HAV1' genes suitable for the nuclear genetic expression of Chlamydomonas reinhardtii, as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; The HAV2 and HAV2' genes are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively; HAV3 and HAV3' genes suitable for the expression of Chlamydomonas reinhardtii chloroplasts are optimized, as shown in SEQ ID NO.5 and SEQ ID NO. .6 shown. The above codon-optimized avian influenza antigen genes SEQ ID NO.1-SEQ ID NO.6 were artificially synthesized by Sangon Bioengineering (Shangh...
Embodiment 2
[0075] [Example 2] Selection and cultivation of transgenic recipient algal strains
[0076] The selected transgenic recipient algal strains in the present invention are: cell wall-deficient Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, cc-849, purchased from the Chlamydomonas Genetics Center of Duck University, USA) and cytochrome b-deficient Chlamydomonas reinhardtii (cc-2654, purchased from the United States) from the Chlamydomonas Genetics Center at Duck University, USA). TAP was selected as the medium for the transgenic algal strain, and its preparation method was as follows: 25mL of 4×Beijerinck salts (4g MgSO 4 .7H 2 O, 2gCaCl 2 .2H 2 O, 16g NH 4 Dissolve Cl in deionized water, make up to 1L), 2.42g Tris, 1mL 1M(K)PO 4 , 1mL Trace trace element mixture (50g Na 2 EDTA, 1.1g (NH 4 ) 6 Mo 7 O 24 , 1.57gCuSO 4 .5H 2 O, 1.61g CoCl 2 .6H 2 O, 4.99g FeSO 4 .7H 2 O, 22g ZnSO 4 .7H 2 O, 5.6gMnCl 2 .4H 2 O, 11.4g H 3 BO 3 Dilute to 1L deionized water)...
Embodiment 3
[0077] [Example 3] Construction of a genetic expression vector for transgenic avian influenza antigen gene Chlamydomonas reinhardtii
[0078] (1) Construction of avian influenza antigen gene Chlamydomonas reinhardtii nuclear genetic expression vector
[0079] It can be seen from Example 1 that the plasmid pUC57HAV1 contains the HAV1 gene, and pUC57HAV1' contains the HAV1' gene. Since the appropriate restriction enzyme sites were added while optimizing the sequence, the restriction enzymes Nhe I and Pmac Ⅰ Double enzyme digestion (purchased from Fermentas) to obtain HAV1 and HAV1' genes. Plasmid pH105 (Wu JX et al. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii. Chinese Journal of Oceanology and Limnology. Vol.26No.3, P.242-247, 2008 was digested with EcoRⅠ ) to obtain a portion of about 731 bp containing the HSP70A-RBCS2 promoter and RBCS2 terminator, which was inserted into pSP124 (Lambreras v, Stevens DR,...
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